Team:OUC-China/Result/date

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<div><p>We've also shared this Data document on Google Doc, <a href="https://docs.google.com/document/d/106boR2c3uczJ1yOioUxMxTKYvC9hDI8_1T_9dwrY0QA/edit">see here.</a></p></div>
<div><p class="c2 c22"><span></span></p></div><h1 class="c4"><a name="h.u9me3k1zft2"></a><span>7.21</span></h1><h3 class="c4"><a name="h.5c3btudrepao"></a><span>I &nbsp;Prepare medium</span></h3><h3 class="c4"><a name="h.5c3btudrepao"></a><span>1.soc medium</span></h3><h3 class="c4"><a name="h.5c3btudrepao"></a><span>2.LB medium</span></h3><h3 class="c4"><a name="h.5c3btudrepao"></a><span>3.LBliquid medium</span></h3><h3 class="c4"><a name="h.5c3btudrepao"></a><span>II &nbsp;transform 2011 parts &ndash;OUC Order 1 to 3</span></h3><h3 class="c4"><a name="h.5c3btudrepao"></a><span>III &nbsp;pick up a tested single colony</span></h3><p class="c2"><span></span></p><h1 class="c4"><a name="h.tutvix3ykf10"></a><span>7.22</span></h1><h3 class="c4"><a name="h.g2bioc2tx8l7"></a><span>1&#65294;Extract plasmid of the practicing part</span></h3><h3 class="c4"><a name="h.ufnfesh49wep"></a><span>2. Measure density using distilled water as comparison</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c40"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>A260/A280</span></p></td><td class="c13"><p class="c4"><span>A260/A230</span></p></td><td class="c13"><p class="c4"><span>density&#65288;ng/ul&#65289;</span></p></td></tr><tr class="c40"><td class="c5"><p class="c4"><span>1</span></p></td><td class="c5"><p class="c4"><span>1.892</span></p></td><td class="c13"><p class="c4"><span>2.115</span></p></td><td class="c13"><p class="c4"><span>0.035</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>2</span></p></td><td class="c5"><p class="c4"><span>1.841</span></p></td><td class="c13"><p class="c4"><span>1.227</span></p></td><td class="c13"><p class="c4"><span>0.0405</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>3</span></p></td><td class="c5"><p class="c4"><span>2.299</span></p></td><td class="c13"><p class="c4"><span>4.054</span></p></td><td class="c13"><p class="c4"><span>0.030</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.9fgyukkhs06"></a><span>3. Digestion for 3 hours</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c42"><p class="c2"><span></span></p><p class="c2"><span></span></p></td><td class="c32"><p class="c4"><span>1</span></p></td><td class="c32"><p class="c4"><span>2</span></p></td><td class="c32"><p class="c4"><span>3</span></p></td><td class="c32"><p class="c4"><span>4(Single enzyme cut)</span></p><p class="c2"><span></span></p></td></tr><tr class="c12"><td class="c42"><p class="c4"><span>DNA(ul)</span></p></td><td class="c32"><p class="c4"><span>15</span></p></td><td class="c32"><p class="c4"><span>15</span></p></td><td class="c32"><p class="c4"><span>15</span></p></td><td class="c32"><p class="c4"><span>15</span></p></td></tr><tr class="c12"><td class="c42"><p class="c4"><span>Buffer2(ul)</span></p></td><td class="c32"><p class="c4"><span>5</span></p></td><td class="c32"><p class="c4"><span>5</span></p></td><td class="c32"><p class="c4"><span>5</span></p></td><td class="c32"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c42"><p class="c4"><span>Bsa(ul)</span></p></td><td class="c32"><p class="c4"><span>0.5</span></p></td><td class="c32"><p class="c4"><span>0.5</span></p></td><td class="c32"><p class="c4"><span>0.5</span></p></td><td class="c32"><p class="c4"><span>-----</span></p></td></tr><tr class="c12"><td class="c42"><p class="c4"><span>Double-distilled water(ul)</span></p></td><td class="c32"><p class="c4"><span>27.5</span></p></td><td class="c32"><p class="c4"><span>27.5</span></p></td><td class="c32"><p class="c4"><span>27.5</span></p></td><td class="c32"><p class="c4"><span>29</span></p></td></tr><tr class="c12"><td class="c42"><p class="c4"><span>EcoR I(ul)</span></p></td><td class="c32"><p class="c4"><span>1</span></p></td><td class="c32"><p class="c4"><span>1</span></p></td><td class="c32"><p class="c4"><span>1</span></p></td><td class="c32"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c42"><p class="c4"><span>Pst I(ul)</span></p></td><td class="c32"><p class="c4"><span>1</span></p></td><td class="c32"><p class="c4"><span>1</span></p></td><td class="c32"><p class="c4"><span>1</span></p></td><td class="c32"><p class="c4"><span>-------</span></p></td></tr><tr class="c12"><td class="c42"><p class="c4"><span>Time(h)</span></p></td><td class="c32"><p class="c4"><span>3</span></p></td><td class="c32"><p class="c4"><span>3</span></p></td><td class="c32"><p class="c4"><span>3</span></p></td><td class="c32"><p class="c4"><span>3</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.1ruf9mihj3z2"></a><span>4.check by Electrophoresis</span></h3><p class="c4"><span class="c27">1&#12289;2&#12289;3&#65306;double enzyme&#65292;4&#12289;5&#12289;6&#65306;Single enzyme &#65292;7&#65306;plasmid</span></p><p class="c4"><span class="c27">5.transform 7new parts(number:4-10)</span></p><p class="c4"><span class="c27">6.culture bacteria on LB medium</span></p><p class="c4"><span class="c27">7.culture 3 new parts in Shaker</span></p><h1 class="c4"><a name="h.90bsowdqfwvf"></a><span>7.24</span></h1><h3 class="c4"><a name="h.fllej6viiwbz"></a><span>1. Presrvation species of parts No. ,4,5,6,7,8,9,10</span></h3><h3 class="c4"><a name="h.fllej6viiwbz"></a><span>2. Extract plasmid of 4,5,6,7,8,9,10</span></h3><h3 class="c4"><a name="h.fllej6viiwbz"></a><span>3.check through double enzyme Digestion</span></h3><h3 class="c4 c29"><a name="h.fllej6viiwbz"></a></h3><h1 class="c4"><a name="h.60w0ipkcfych"></a><span>7.25</span></h1><h3 class="c4"><a name="h.r7rj1zpq27u6"></a><span>1. Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c37"><p class="c4"><span>5</span></p></td><td class="c37"><p class="c4"><span>0.020&micro;g/&micro;l</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>4</span></p></td><td class="c37"><p class="c4"><span>0.012&micro;g/&micro;l</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>6</span></p></td><td class="c37"><p class="c4"><span>0.020&micro;g/&micro;l</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>2</span></p></td><td class="c37"><p class="c4"><span>0.015&micro;g/&micro;l</span></p></td></tr></tbody></table><p class="c2"><span></span></p><p class="c2"><span></span></p><h3 class="c4"><a name="h.kj1r3fgjzq4o"></a><span>2. Digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>DNA</span></p></td><td class="c1"><p class="c4"><span>25</span></p></td><td class="c1"><p class="c4"><span>30</span></p></td><td class="c1"><p class="c4"><span>25</span></p></td><td class="c1"><p class="c4"><span>25</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Buffer2</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>BSA</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>H2O</span></p></td><td class="c1"><p class="c4"><span>17.5</span></p></td><td class="c1"><p class="c4"><span>12.5</span></p></td><td class="c1"><p class="c4"><span>17.5</span></p></td><td class="c1"><p class="c4"><span>17.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>enzyme1</span></p></td><td class="c1"><p class="c4"><span>EcoR&#8544;1&micro;l</span></p></td><td class="c1"><p class="c4"><span>EcoR&#8544;1&micro;l</span></p></td><td class="c1"><p class="c4"><span>Spe&#8544;1&micro;l</span></p></td><td class="c1"><p class="c4"><span>Xba&#8544;1&micro;l</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Enzyme2</span></p></td><td class="c1"><p class="c4"><span>Spe&#8544;1&micro;l</span></p></td><td class="c1"><p class="c4"><span>Xba&#8544;1&micro;l</span></p></td><td class="c1"><p class="c4"><span>Pst&#8544;1&micro;l</span></p></td><td class="c1"><p class="c4"><span>Pst&#8544;1&micro;l</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.z9jf6h5q8571"></a><span>3.check by Electrophoresis</span></h3><h3 class="c4"><a name="h.z9jf6h5q8571"></a><span>4.gel extraction</span></h3><h3 class="c4"><a name="h.3xuy6enwwjqe"></a><span>5.measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c4"><span>OUC-order</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Density &nbsp;ug/&micro;l</span></p></td><td class="c1"><p class="c4"><span>0.016</span></p></td><td class="c1"><p class="c4"><span>0.021</span></p></td><td class="c1"><p class="c4"><span>0.027</span></p></td><td class="c1"><p class="c4"><span>0.028</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.7t6dpq2z3uet"></a><span>6. Ligate overnight</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Parts</span></p></td><td class="c9"><p class="c4"><span>2&micro;l 5+3&micro;l 4</span></p></td><td class="c9"><p class="c4"><span>3&micro;l 2+3&micro;l 6</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer</span></p></td><td class="c9"><p class="c4"><span>2&micro;l</span></p></td><td class="c9"><p class="c4"><span>2&micro;l</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>enzyme</span></p></td><td class="c9"><p class="c4"><span>1&micro;l</span></p></td><td class="c9"><p class="c4"><span>1&micro;l</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DdH2O</span></p></td><td class="c9"><p class="c4"><span>2&micro;l</span></p></td><td class="c9"><p class="c4"><span>1&micro;l</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.v8m6lz1ufk1s"></a><span>7.28</span></h1><h3 class="c4"><a name="h.3yqybicztq70"></a><span>Repeat the ligation expriments of 7.25</span></h3><h1 class="c4"><a name="h.iezpb3pvvzxm"></a><span>7.29</span></h1><h3 class="c4"><a name="h.2tk3jc38wcfz"></a><span>Extract plasmids of parts No. 11,12,13,14</span></h3><h1 class="c4"><a name="h.igfocjjyx4ru"></a><span>7.30</span></h1><h3 class="c4"><a name="h.i6nzwpfyvvjl"></a><span>Medium has antibiotic resistance problem, re-transform 11\12\13\14</span></h3><h1 class="c4"><a name="h.ug8l8jxvj3n1"></a><span>7.31</span></h1><h3 class="c4"><a name="h.2qt7wbgkde7s"></a><span>1.cultrue bacteria of 11,12,13,14 in LB liquid medium</span></h3><h3 class="c4"><a name="h.2qt7wbgkde7s"></a><span>2. Extract plasmid of 15,16,17,18,19,20,21,22</span></h3><h3 class="c4"><a name="h.2qt7wbgkde7s"></a><span>3. Optimization the Steps of Extracting plasmid</span></h3><h3 class="c4"><a name="h.2qt7wbgkde7s"></a><span>4.Chloramphericol preparing</span></h3><h3 class="c4"><a name="h.2qt7wbgkde7s"></a><span>5. NaAC&rsquo;s DNA </span><span><a class="c45" href="http:///t">concentration</a></span></h3><p class="c2"><span><a class="c45" href="http:///t"></a></span></p><h1 class="c4"><a name="h.tfyl7y9pxkmo"></a><span>8.1 </span></h1><h3 class="c4"><a name="h.bx2edw2baey7"></a><span>1. Extract plasmid of 11-14 and Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>ng/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>11</span></p></td><td class="c9"><p class="c4"><span>12.7</span></p></td><td class="c9"><p class="c4"><span>1.917</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>12</span></p></td><td class="c9"><p class="c4"><span>10.3</span></p></td><td class="c9"><p class="c4"><span>1.981</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>13</span></p></td><td class="c9"><p class="c4"><span>35.5</span></p></td><td class="c9"><p class="c4"><span>1.919</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>14</span></p></td><td class="c9"><p class="c4"><span>15.7</span></p></td><td class="c9"><p class="c4"><span>1.975</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.hvilmpnl4guw"></a><span>2. Extract plasmid of No.5,4,6,2&#65292;15-20 parts</span></h3><h3 class="c4"><a name="h.hvilmpnl4guw"></a><span>3.culture the colony of GFP on LB medium</span></h3><h1 class="c4"><a name="h.9dt18ihmbdii"></a><span>8.2</span></h1><h3 class="c4"><a name="h.e15sf2yo8hvd"></a><span>Measure density of 15-22,2,4,5,6</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>Parts</span></p></td><td class="c9"><p class="c4"><span>&micro;g/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>0.038</span></p></td><td class="c9"><p class="c4"><span>1.875</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>4</span></p></td><td class="c9"><p class="c4"><span>0.019</span></p></td><td class="c9"><p class="c4"><span>1.298</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>5</span></p></td><td class="c9"><p class="c4"><span>0.022</span></p></td><td class="c9"><p class="c4"><span>1.538</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>6</span></p></td><td class="c9"><p class="c4"><span>0.020</span></p></td><td class="c9"><p class="c4"><span>1.702</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>15</span></p></td><td class="c9"><p class="c4"><span>0.012</span></p></td><td class="c9"><p class="c4"><span>1.237</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>16</span></p></td><td class="c9"><p class="c4"><span>0.024</span></p></td><td class="c9"><p class="c4"><span>1.649</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>17</span></p></td><td class="c9"><p class="c4"><span>0.018</span></p></td><td class="c9"><p class="c4"><span>1.731</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18</span></p></td><td class="c9"><p class="c4"><span>0.015</span></p></td><td class="c9"><p class="c4"><span>1.470</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>21</span></p></td><td class="c9"><p class="c4"><span>0.017</span></p></td><td class="c9"><p class="c4"><span>1.538</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.i1axlh3f3s5k"></a><span>8.3</span></h1><h3 class="c4"><a name="h.q8al8jclaxs9"></a><span>1. Concentrate plasmid using alcohol and NaAC</span></h3><h3 class="c4"><a name="h.q8al8jclaxs9"></a><span>2. Measure density after concentrating</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>ng/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>103</span></p></td><td class="c9"><p class="c4"><span>1.807</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>4</span></p></td><td class="c9"><p class="c4"><span>33</span></p></td><td class="c9"><p class="c4"><span>1.811</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>5</span></p></td><td class="c9"><p class="c4"><span>56</span></p></td><td class="c9"><p class="c4"><span>1.836</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>6</span></p></td><td class="c9"><p class="c4"><span>41</span></p></td><td class="c9"><p class="c4"><span>1.745</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.s9gc90q0jnk2"></a><span>8.4</span></h1><h3 class="c4"><a name="h.w2vf5a97h188"></a><span>Digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>Double enzymes</span></p></td><td class="c1"><p><span></span></p></td><td class="c1"><p class="c4"><span>Single enzyme</span></p></td><td class="c1"><p><span></span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Parts</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>6</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>DNA</span></p></td><td class="c1"><p class="c4"><span>25</span></p></td><td class="c1"><p class="c4"><span>13</span></p></td><td class="c1"><p class="c4"><span>27</span></p></td><td class="c1"><p class="c4"><span>25</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Buffer</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>BSA</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>ddH2O</span></p></td><td class="c1"><p class="c4"><span>16.5</span></p></td><td class="c1"><p class="c4"><span>28.5</span></p></td><td class="c1"><p class="c4"><span>16.5</span></p></td><td class="c1"><p class="c4"><span>18.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>enzyme</span></p></td><td class="c1"><p class="c4"><span>EcoR&#8544; 1.5</span></p></td><td class="c1"><p class="c4"><span>Xba&#8544;1.5</span></p></td><td class="c1"><p class="c4"><span>Xba&#8544;1</span></p></td><td class="c1"><p class="c4"><span>Spe&#8544;1</span></p></td></tr><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>Spe&#8544;1.5</span></p></td><td class="c1"><p class="c4"><span>Pst&#8544;1.5</span></p></td><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c2"><span></span></p></td></tr></tbody></table><p class="c2"><span></span></p><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>Parts</span></p></td><td class="c9"><p class="c4"><span>4</span></p></td><td class="c9"><p class="c4"><span>6</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>28.5</span></p></td><td class="c9"><p class="c4"><span>30</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer</span></p></td><td class="c9"><p class="c4"><span>EcoR&#8544;buffer 5</span></p></td><td class="c9"><p class="c4"><span>Buffer3 5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>BSA</span></p></td><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>15.5</span></p></td><td class="c9"><p class="c4"><span>13.5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>enzyme</span></p></td><td class="c9"><p class="c4"><span>EcoR&#8544;1</span></p></td><td class="c9"><p class="c4"><span>Pst&#8544;1</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.hhwrahhkoe43"></a><span>8.5</span></h1><h3 class="c4"><a name="h.t0nrpb7qcvp0"></a><span>1. Begin device 2</span></h3><h3 class="c4"><a name="h.t0nrpb7qcvp0"></a><span>2. Design experimental procedures: </span></h3><h3 class="c4"><a name="h.t0nrpb7qcvp0"></a><span>3. First construction with parts of device 2</span></h3><p class="c2"><span class="c44"></span></p><h1 class="c4"><a name="h.tzzxaggc2un4"></a><span>8.6</span></h1><h3 class="c4"><a name="h.yzze9tnt5vuy"></a><span>2+6 successfully ligate</span></h3><h3 class="c4"><a name="h.8c49qiwztmgq"></a><span>Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>Parts</span></p></td><td class="c9"><p class="c4"><span>&micro;g/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>0.085</span></p></td><td class="c9"><p class="c4"><span>1.910</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>16</span></p></td><td class="c9"><p class="c4"><span>0.119</span></p></td><td class="c9"><p class="c4"><span>1.867</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18</span></p></td><td class="c9"><p class="c4"><span>0.019</span></p></td><td class="c9"><p class="c4"><span>2.033</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.nzti3vckiivz"></a><span>Measure density after concentrating</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>Parts</span></p></td><td class="c9"><p class="c4"><span>&micro;g/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18</span></p></td><td class="c9"><p class="c4"><span>0.0605</span></p></td><td class="c9"><p class="c4"><span>1.921</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>11</span></p></td><td class="c9"><p class="c4"><span>0.0335</span></p></td><td class="c9"><p class="c4"><span>2.107</span></p></td></tr></tbody></table><h3 class="c29 c4"><a name="h.ewnnzrlm06qu"></a></h3><h3 class="c4"><a name="h.pf64q93rtmr3"></a><span>Digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>Double enzymes</span></p></td><td class="c1"><p><span></span></p></td><td class="c1"><p class="c4"><span>Single enzyme</span></p></td><td class="c1"><p><span></span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Parts</span></p></td><td class="c1"><p class="c4"><span>18</span></p></td><td class="c1"><p class="c4"><span>16</span></p></td><td class="c1"><p class="c4"><span>3</span></p></td><td class="c1"><p class="c4"><span>11</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>DNA</span></p></td><td class="c1"><p class="c4"><span>20</span></p></td><td class="c1"><p class="c4"><span>10</span></p></td><td class="c1"><p class="c4"><span>15</span></p></td><td class="c1"><p class="c4"><span>29</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Buffer</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>BSA</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>ddH2O</span></p></td><td class="c1"><p class="c4"><span>21.5</span></p></td><td class="c1"><p class="c4"><span>31.5</span></p></td><td class="c1"><p class="c4"><span>28</span></p></td><td class="c1"><p class="c4"><span>14.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>enzyme</span></p></td><td class="c1"><p class="c4"><span>EcoR&#8544; 1.5</span></p></td><td class="c1"><p class="c4"><span>Xba&#8544;1.5</span></p></td><td class="c1"><p class="c4"><span>Xba&#8544;1</span></p></td><td class="c1"><p class="c4"><span>Spe&#8544;1</span></p></td></tr><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>Spe&#8544;1.5</span></p></td><td class="c1"><p class="c4"><span>Pst&#8544;1.5</span></p></td><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c2"><span></span></p></td></tr></tbody></table><h3 class="c4"><a name="h.id7koc7n89jl"></a><span>After 8hours </span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><h3 class="c4"><a name="h.euom5tw4uac7"></a><span>Parts</span></h3></td><td class="c9"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>11</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>20</span></p></td><td class="c9"><p class="c4"><span>20</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer</span></p></td><td class="c9"><p class="c4"><span>EcoR&#8544;buffer 5</span></p></td><td class="c9"><p class="c4"><span>Buffer3 5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>BSA</span></p></td><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>23.5</span></p></td><td class="c9"><p class="c4"><span>23</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>enzyme</span></p></td><td class="c9"><p class="c4"><span>EcoR&#8544;1</span></p></td><td class="c9"><p class="c4"><span>Pst&#8544;1</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.ekhxnmiid85t"></a><span>8.7</span></h1><h3 class="c4"><a name="h.zb49mmwonyx9"></a><span>1. Checking 4+5, 2+6 &lsquo;s ligation by Gel electrophoresis</span></h3><h3 class="c4"><a name="h.sts8pafwtw56"></a><span>&nbsp;Digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>&mu;L</span></p></td><td class="c9"><p class="c4"><span>2+6</span></p></td><td class="c9"><p class="c4"><span>4+5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>2&#65288;53ng&#65289;</span></p></td><td class="c9"><p class="c4"><span>3(28ng)</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer2</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>BSA</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>6.7</span></p></td><td class="c9"><p class="c4"><span>5.9</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>EcoR&#8544;</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Pst&#8544;</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td></tr></tbody></table><h3 class="c29 c4"><a name="h.7hb0wq9fet7i"></a></h3><h3 class="c4"><a name="h.pxbfcxpcw2h0"></a><span>check by small gel</span></h3><h1 class="c49 c4"><a name="h.1n6cx51ccc1m"></a></h1><h1 class="c4"><a name="h.hzg362nyf3j"></a><span>8.8</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.ms09fpm4vjum"></a><span>1.transform the parts of 3-18,11-16,14-19</span></h3><h3 class="c4"><a name="h.ms09fpm4vjum"></a><span>2. Active bacteria of 4.5.18.11 and culture in LB liquid medium</span></h3><h3 class="c4"><a name="h.ms09fpm4vjum"></a><span>3. Presrvation species parts of 4-5</span></h3><p class="c2"><span></span></p><h1 class="c4"><a name="h.fel77li0jvq3"></a><span>8.9</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.golezolfnu2v"></a><span>1.check 2+6,4+5 by small gel </span></h3><h3 class="c4"><a name="h.golezolfnu2v"></a><span>2. Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>number</span></p></td><td class="c13"><p class="c4"><span>ng/&micro;l</span></p></td><td class="c13"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>2+6</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>58</span></p></td><td class="c13"><p class="c4"><span>1.902</span></p></td></tr><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>78.5</span></p></td><td class="c13"><p class="c4"><span>1.826</span></p></td></tr><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>3</span></p></td><td class="c13"><p class="c4"><span>62.5</span></p></td><td class="c13"><p class="c4"><span>1.953</span></p></td></tr><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>4</span></p></td><td class="c13"><p class="c4"><span>60.5</span></p></td><td class="c13"><p class="c4"><span>2.017</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>4+5</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>31</span></p></td><td class="c13"><p class="c4"><span>2.081</span></p></td></tr><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>33</span></p></td><td class="c13"><p class="c4"><span>2.089</span></p></td></tr><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>3</span></p></td><td class="c13"><p class="c4"><span>20.5</span></p></td><td class="c13"><p class="c4"><span>2.291</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.95qz8jl117p"></a><span>3. Digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c10"><p class="c4"><span>parts</span></p></td><td class="c10"><p class="c4"><span>2+6</span></p></td><td class="c10"><p><span></span></p></td><td class="c10"><p><span></span></p></td><td class="c10"><p><span></span></p></td><td class="c10"><p class="c4"><span>5+4</span></p></td><td class="c16"><p><span></span></p></td><td class="c16"><p><span></span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>number</span></p></td><td class="c10"><p class="c4"><span>1</span></p></td><td class="c10"><p class="c4"><span>2</span></p></td><td class="c10"><p class="c4"><span>3</span></p></td><td class="c10"><p class="c4"><span>4</span></p></td><td class="c10"><p class="c4"><span>1</span></p></td><td class="c16"><p class="c4"><span>2</span></p></td><td class="c16"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>DNA</span></p></td><td class="c10"><p class="c4"><span>2</span></p></td><td class="c10"><p class="c4"><span>2</span></p></td><td class="c10"><p class="c4"><span>2</span></p></td><td class="c10"><p class="c4"><span>2</span></p></td><td class="c10"><p class="c4"><span>3</span></p></td><td class="c16"><p class="c4"><span>3</span></p></td><td class="c16"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>Buffer3</span></p></td><td class="c10"><p class="c4"><span>1</span></p></td><td class="c10"><p class="c4"><span>1</span></p></td><td class="c10"><p class="c4"><span>1</span></p></td><td class="c10"><p class="c4"><span>1</span></p></td><td class="c10"><p class="c4"><span>1</span></p></td><td class="c16"><p class="c4"><span>1</span></p></td><td class="c16"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>BSA</span></p></td><td class="c10"><p class="c4"><span>0.1</span></p></td><td class="c10"><p class="c4"><span>0.1</span></p></td><td class="c10"><p class="c4"><span>0.1</span></p></td><td class="c10"><p class="c4"><span>0.1</span></p></td><td class="c10"><p class="c4"><span>0.1</span></p></td><td class="c16"><p class="c4"><span>0.1</span></p></td><td class="c16"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>ddH2O</span></p></td><td class="c10"><p class="c4"><span>6.5</span></p></td><td class="c10"><p class="c4"><span>6.5</span></p></td><td class="c10"><p class="c4"><span>6.5</span></p></td><td class="c10"><p class="c4"><span>6.5</span></p></td><td class="c10"><p class="c4"><span>5.5</span></p></td><td class="c16"><p class="c4"><span>5.5</span></p></td><td class="c16"><p class="c4"><span>5.5</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>EcoR&#8544;</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c16"><p class="c4"><span>0.2</span></p></td><td class="c16"><p class="c4"><span>0.2</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>Pst&#8544;</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c16"><p class="c4"><span>0.2</span></p></td><td class="c16"><p class="c4"><span>0.2</span></p></td></tr></tbody></table><h3 class="c29 c4"><a name="h.ew5rjvlhlbes"></a></h3><h3 class="c4"><a name="h.ew5rjvlhlbes"></a><span>4. Extract plasmid of &nbsp;4,5,18,11</span></h3><h3 class="c4"><a name="h.fbuct55jz3p2"></a><span>Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><h3 class="c4"><a name="h.hqsj9u31i72y"></a><span>number</span></h3></td><td class="c9"><p class="c4"><span>ng/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>5</span></p></td><td class="c9"><p class="c4"><span>73</span></p></td><td class="c9"><p class="c4"><span>2.000</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>4</span></p></td><td class="c9"><p class="c4"><span>71.5</span></p></td><td class="c9"><p class="c4"><span>2.072</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18</span></p></td><td class="c9"><p class="c4"><span>32.5</span></p></td><td class="c9"><p class="c4"><span>2.305</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>85</span></p></td><td class="c9"><p class="c4"><span>1.910</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>11</span></p></td><td class="c9"><p class="c4"><span>95.5</span></p></td><td class="c9"><p class="c4"><span>1.950</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>16</span></p></td><td class="c9"><p class="c4"><span>119</span></p></td><td class="c9"><p class="c4"><span>1.867</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.3z29ux1deqzo"></a><span>Digestion 50&micro;l</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c17"><p class="c2"><span></span></p></td><td class="c17"><p class="c4"><span>5</span></p></td><td class="c21"><p class="c4"><span>4</span></p></td><td class="c17"><p class="c4"><span>18</span></p></td><td class="c21"><p class="c4"><span>3</span></p></td><td class="c21"><p class="c4"><span>11</span></p></td><td class="c21"><p class="c4"><span>16</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>DNA</span></p></td><td class="c17"><p class="c4"><span>13</span></p></td><td class="c21"><p class="c4"><span>13</span></p></td><td class="c17"><p class="c4"><span>30</span></p></td><td class="c21"><p class="c4"><span>11</span></p></td><td class="c21"><p class="c4"><span>10</span></p></td><td class="c21"><p class="c4"><span>8</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>Buffer3</span></p></td><td class="c17"><p class="c4"><span>5</span></p></td><td class="c21"><p class="c4"><span>5</span></p></td><td class="c17"><p class="c4"><span>5</span></p></td><td class="c21"><p class="c4"><span>5</span></p></td><td class="c21"><p class="c4"><span>5</span></p></td><td class="c21"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>BSA</span></p></td><td class="c17"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td><td class="c17"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>ddH2O</span></p></td><td class="c17"><p class="c4"><span>29.5</span></p></td><td class="c21"><p class="c4"><span>29.5</span></p></td><td class="c17"><p class="c4"><span>19.5</span></p></td><td class="c21"><p class="c4"><span>31.5</span></p></td><td class="c21"><p class="c4"><span>30.5</span></p></td><td class="c21"><p class="c4"><span>34.5</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>enzyme</span></p></td><td class="c17"><p class="c4"><span>EcoR&#8544; 1</span></p></td><td class="c21"><p class="c4"><span>Xba&#8544;1</span></p></td><td class="c17"><p class="c4"><span>EcoR&#8544; 1</span></p></td><td class="c21"><p class="c4"><span>Xba&#8544;1</span></p></td><td class="c21"><p class="c4"><span>EcoR&#8544; 1</span></p></td><td class="c21"><p class="c4"><span>Xba&#8544;1</span></p></td></tr><tr class="c12"><td class="c17"><p class="c2"><span></span></p></td><td class="c17"><p class="c4"><span>Spe&#8544;1</span></p></td><td class="c21"><p class="c4"><span>Pst&#8544;1</span></p></td><td class="c17"><p class="c4"><span>Spe&#8544;1</span></p></td><td class="c21"><p class="c4"><span>Pst&#8544;1</span></p></td><td class="c21"><p class="c4"><span>Spe&#8544;1</span></p></td><td class="c21"><p class="c4"><span>Pst&#8544;1</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.jmqfpq9xkm9z"></a><span>8.11</span></h1><h4 class="c4"><a name="h.m6rgdwzdsd3p"></a><span>1. Checking ligation correctness of 14&amp;19 using small gel----fails</span></h4><h4 class="c4"><a name="h.m6rgdwzdsd3p"></a><span>2.try to ligate using 3A</span></h4><h1 class="c4"><a name="h.z0x6hx6e7ltc"></a><span>8.12</span></h1><h3 class="c4"><a name="h.dm8f9683o5dr"></a><span>1. Extract plasmid of &nbsp;5+4 and Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span>ng/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>55</span></p></td><td class="c9"><p class="c4"><span>1.964</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>39</span></p></td><td class="c9"><p class="c4"><span>2.324</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>41</span></p></td><td class="c9"><p class="c4"><span>2.412</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>4</span></p></td><td class="c9"><p class="c4"><span>81</span></p></td><td class="c9"><p class="c4"><span>2.051</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>5</span></p></td><td class="c9"><p class="c4"><span>42</span></p></td><td class="c9"><p class="c4"><span>2.154</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>6</span></p></td><td class="c9"><p class="c4"><span>58</span></p></td><td class="c9"><p class="c4"><span>1.966</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.77mylrq8vnt2"></a><span>2.check by single enzyme digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c17"><p class="c4"><span>number</span></p></td><td class="c17"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>2</span></p></td><td class="c17"><p class="c4"><span>3</span></p></td><td class="c21"><p class="c4"><span>4</span></p></td><td class="c21"><p class="c4"><span>5</span></p></td><td class="c21"><p class="c4"><span>6</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>DNA</span></p></td><td class="c17"><p class="c4"><span>3</span></p></td><td class="c21"><p class="c4"><span>4</span></p></td><td class="c17"><p class="c4"><span>4</span></p></td><td class="c21"><p class="c4"><span>2</span></p></td><td class="c21"><p class="c4"><span>4</span></p></td><td class="c21"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>Buffer EcoR I</span></p></td><td class="c17"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c17"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>ddH2O</span></p></td><td class="c17"><p class="c4"><span>5.5</span></p></td><td class="c21"><p class="c4"><span>4.5</span></p></td><td class="c17"><p class="c4"><span>4.5</span></p></td><td class="c21"><p class="c4"><span>6.5</span></p></td><td class="c21"><p class="c4"><span>4.5</span></p></td><td class="c21"><p class="c4"><span>5.5</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>EcoR&#8544;</span></p></td><td class="c17"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td><td class="c17"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td></tr></tbody></table><h3 class="c29 c4"><a name="h.sp1o9aob8jry"></a></h3><h3 class="c4"><a name="h.f568ba30zhy7"></a><span>Check using small gel</span></h3><h3 class="c4"><a name="h.f568ba30zhy7"></a><span>3.cultrue bacteria of part 13</span></h3><h3 class="c4"><a name="h.f568ba30zhy7"></a><span>4. ligate &nbsp;2+6+3,11+16,18+13</span></h3><p class="c2"><span></span></p><h1 class="c4"><a name="h.alf83syip56z"></a><span>8.13</span></h1><h3 class="c4"><a name="h.ke3ebgbum5n"></a><span>1.Extract plasmid </span></h3><h3 class="c4"><a name="h.ke3ebgbum5n"></a><span>2.pick colony of 2+6+3,18+3,16+3 and culture in shaker</span></h3><p class="c2"><span></span></p><h1 class="c4"><a name="h.e0ipvrwodbd7"></a><span>8.14 </span></h1><h4 class="c4"><a name="h.qq0p6vx7yo2i"></a><span>Checking whether colony of different size contain different plasmids, or whether could be used to pick colony raising transformation possibility. But it made no difference</span></h4><h1 class="c4"><a name="h.5k95r8qmtjok"></a><span>8.15</span></h1><h3 class="c4"><a name="h.wy8969l7pop"></a><span>1.measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span>&micro;g/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>2+6+3-1</span></p></td><td class="c9"><p class="c4"><span>0.032</span></p></td><td class="c9"><p class="c4"><span>3.439</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>2+6+3-2</span></p></td><td class="c9"><p class="c4"><span>0.064</span></p></td><td class="c9"><p class="c4"><span>2.370</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>11+16</span></p></td><td class="c9"><p class="c4"><span>0.056</span></p></td><td class="c9"><p class="c4"><span>2.306</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>11+16</span></p></td><td class="c9"><p class="c4"><span>0.102</span></p></td><td class="c9"><p class="c4"><span>2.010</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18+3</span></p></td><td class="c9"><p class="c4"><span>0.037</span></p></td><td class="c9"><p class="c4"><span>2.434</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18+3</span></p></td><td class="c9"><p class="c4"><span>0.033</span></p></td><td class="c9"><p class="c4"><span>2.332</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.3m4xfb4u4qv8"></a><span>2. Digestion</span></h3><p class="c2"><span></span></p><h3 class="c4"><a name="h.c1ekew4tc9t5"></a><span>Single enzyme 10&micro;l</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c17"><p class="c4"><span>number</span></p></td><td class="c17"><p class="c4"><span>2+6+3-1</span></p></td><td class="c21"><p class="c4"><span>2+6+3-2</span></p></td><td class="c17"><p class="c4"><span>3+18-1</span></p></td><td class="c21"><p class="c4"><span>3+18-2</span></p></td><td class="c21"><p class="c4"><span>11+16</span></p></td><td class="c21"><p class="c4"><span>11+16</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>DNA</span></p></td><td class="c17"><p class="c4"><span>4</span></p></td><td class="c21"><p class="c4"><span>2</span></p></td><td class="c17"><p class="c4"><span>4</span></p></td><td class="c21"><p class="c4"><span>6</span></p></td><td class="c21"><p class="c4"><span>2.5</span></p></td><td class="c21"><p class="c4"><span>1.5</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>Buffer EcoR I</span></p></td><td class="c17"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c17"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>ddH2O</span></p></td><td class="c17"><p class="c4"><span>4.7</span></p></td><td class="c21"><p class="c4"><span>6.7</span></p></td><td class="c17"><p class="c4"><span>4.7</span></p></td><td class="c21"><p class="c4"><span>2.7</span></p></td><td class="c21"><p class="c4"><span>6.2</span></p></td><td class="c21"><p class="c4"><span>7.2</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>EcoR&#8544;</span></p></td><td class="c17"><p class="c4"><span>0.3</span></p></td><td class="c21"><p class="c4"><span>0.3</span></p></td><td class="c17"><p class="c4"><span>0.3</span></p></td><td class="c21"><p class="c4"><span>0.3</span></p></td><td class="c21"><p class="c4"><span>0.3</span></p></td><td class="c21"><p class="c4"><span>0.3</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.apo7y8k82owr"></a><span>Double enzyme 10&micro;l</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c11"><p class="c4"><span>number</span></p></td><td class="c20"><p class="c4"><span>18+3-1</span></p></td><td class="c11"><p class="c4"><span>18+3-2</span></p></td><td class="c11"><p class="c4"><span>18</span></p></td><td class="c11"><p class="c4"><span>2+6+3-1</span></p></td><td class="c20"><p class="c4"><span>2+6+3-2</span></p></td><td class="c20"><p class="c4"><span>2+6</span></p></td><td class="c20"><p class="c4"><span>11+16</span></p></td><td class="c20"><p class="c4"><span>11+16</span></p></td></tr><tr class="c12"><td class="c11"><p class="c4"><span>DNA</span></p></td><td class="c20"><p class="c4"><span>4</span></p></td><td class="c11"><p class="c4"><span>6</span></p></td><td class="c11"><p class="c4"><span>6</span></p></td><td class="c11"><p class="c4"><span>4</span></p></td><td class="c20"><p class="c4"><span>2</span></p></td><td class="c20"><p class="c4"><span>3</span></p></td><td class="c20"><p class="c4"><span>2.5</span></p></td><td class="c20"><p class="c4"><span>1.5</span></p></td></tr><tr class="c12"><td class="c11"><p class="c4"><span>Buffer3</span></p></td><td class="c20"><p class="c4"><span>1</span></p></td><td class="c11"><p class="c4"><span>1</span></p></td><td class="c11"><p class="c4"><span>1</span></p></td><td class="c11"><p class="c4"><span>1</span></p></td><td class="c20"><p class="c4"><span>1</span></p></td><td class="c20"><p class="c4"><span>1</span></p></td><td class="c20"><p class="c4"><span>1</span></p></td><td class="c20"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c11"><p class="c4"><span>BSA</span></p></td><td class="c20"><p class="c4"><span>0.1</span></p></td><td class="c11"><p class="c4"><span>0.1</span></p></td><td class="c11"><p class="c4"><span>0.1</span></p></td><td class="c11"><p class="c4"><span>0.1</span></p></td><td class="c20"><p class="c4"><span>0.1</span></p></td><td class="c20"><p class="c4"><span>0.1</span></p></td><td class="c20"><p class="c4"><span>0.1</span></p></td><td class="c20"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c11"><p class="c4"><span>ddH2O</span></p></td><td class="c20"><p class="c4"><span>4.3</span></p></td><td class="c11"><p class="c4"><span>2.3</span></p></td><td class="c11"><p class="c4"><span>2.4</span></p></td><td class="c11"><p class="c4"><span>4.3</span></p></td><td class="c20"><p class="c4"><span>6.3</span></p></td><td class="c20"><p class="c4"><span>5.3</span></p></td><td class="c20"><p class="c4"><span>5.8</span></p></td><td class="c20"><p class="c4"><span>6.8</span></p></td></tr><tr class="c12"><td class="c11"><p class="c4"><span>EcoR&#8544;</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c11"><p class="c4"><span>0.3</span></p></td><td class="c11"><p class="c4"><span>0.3</span></p></td><td class="c11"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td></tr><tr class="c12"><td class="c11"><p class="c4"><span>Pst&#8544;</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c11"><p class="c4"><span>0.3</span></p></td><td class="c11"><p class="c4"><span>0.3</span></p></td><td class="c11"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.l40msbsdoxxo"></a><span>8.16</span></h1><h3 class="c4"><a name="h.9a0lm2k3wig9"></a><span>Check yesterdays&rsquo; digestion by gel electrophoresis</span></h3><p class="c4"><img height="573" src="images/image00.jpg" width="573"></p><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c47"><p class="c4 c22"><span>Ladder</span></p></td><td class="c48"><p class="c4 c22"><span>18-3</span></p></td><td class="c47"><p><span></span></p></td><td class="c47"><p class="c4 c22"><span>18-3</span></p></td><td class="c47"><p><span></span></p></td><td class="c48"><p class="c4 c22"><span>18</span></p></td><td class="c48"><p class="c4 c22"><span>6-2-3</span></p></td><td class="c48"><p class="c4 c22"><span>6-2-3</span></p></td><td class="c48"><p class="c4 c22"><span>6-2</span></p></td></tr><tr class="c12"><td class="c47"><p class="c4 c22"><span>100bp plus</span></p></td><td class="c48"><p class="c4 c22"><span>single</span></p></td><td class="c47"><p class="c4 c22"><span>double</span></p></td><td class="c47"><p class="c4 c22"><span>single</span></p></td><td class="c47"><p class="c4 c22"><span>double</span></p></td><td class="c48"><p class="c4 c22"><span>double</span></p></td><td class="c48"><p class="c4 c22"><span>double</span></p></td><td class="c48"><p class="c4 c22"><span>double</span></p></td><td class="c48"><p class="c4 c22"><span>double</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.6vm4ek23k6ec"></a><span>8.17</span></h1><h3 class="c4"><a name="h.t0sbfske940j"></a><span>1. Presrvation species of pSB1AC3_BBa13017</span></h3><h3 class="c4"><a name="h.n83ej944zlf8"></a><span>2. Extract plasmid of 18&#65292;pSB1AC3_BBa13017&#65292;measure density </span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span>&micro;g/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-1</span></p></td><td class="c9"><p class="c4"><span>0.199</span></p></td><td class="c9"><p class="c4"><span>2.036</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-2</span></p></td><td class="c9"><p class="c4"><span>0.168</span></p></td><td class="c9"><p class="c4"><span>2.036</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-3</span></p></td><td class="c9"><p class="c4"><span>0.113</span></p></td><td class="c9"><p class="c4"><span>2.083</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-4</span></p></td><td class="c9"><p class="c4"><span>0.103</span></p></td><td class="c9"><p class="c4"><span>2.102</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-5</span></p></td><td class="c9"><p class="c4"><span>0.174</span></p></td><td class="c9"><p class="c4"><span>2.129</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-6</span></p></td><td class="c9"><p class="c4"><span>0.147</span></p></td><td class="c9"><p class="c4"><span>2.115</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-7</span></p></td><td class="c9"><p class="c4"><span>0.064</span></p></td><td class="c9"><p class="c4"><span>2.345</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-8</span></p></td><td class="c9"><p class="c4"><span>0.106</span></p></td><td class="c9"><p class="c4"><span>2.186</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-9</span></p></td><td class="c9"><p class="c4"><span>0.163</span></p></td><td class="c9"><p class="c4"><span>2.138</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-10</span></p></td><td class="c9"><p class="c4"><span>0.170</span></p></td><td class="c9"><p class="c4"><span>2.092</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18-1</span></p></td><td class="c9"><p class="c4"><span>0.110</span></p></td><td class="c9"><p class="c4"><span>2.095</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18-2</span></p></td><td class="c9"><p class="c4"><span>0.124</span></p></td><td class="c9"><p class="c4"><span>2.102</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18-3</span></p></td><td class="c9"><p class="c4"><span>0.140</span></p></td><td class="c9"><p class="c4"><span>2.121</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.h74qm3ioftn6"></a><span>3. Prepare medium &nbsp;of &nbsp;TY&#65288;culturing rhizobium&#65289;,CM&#65288;supplemented medium&#65289;</span></h3><h3 class="c4"><a name="h.h74qm3ioftn6"></a><span>4. Digest pSB1AC3_BBa13017 as Carrier</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c37"><p class="c4"><span>DNA</span></p></td><td class="c37"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>Buffer2</span></p></td><td class="c37"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>BSA</span></p></td><td class="c37"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>ddH2O</span></p></td><td class="c37"><p class="c4"><span>37.5</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>EcoR&#8544;</span></p></td><td class="c37"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>Pst&#8544;</span></p></td><td class="c37"><p class="c4"><span>1</span></p></td></tr></tbody></table><p class="c4"><img height="610" src="images/image01.jpg" width="610"></p><h6 class="c4"><a name="h.jnl1gq2qm9xp"></a><span>Extract 3055bp strip</span></h6><h1 class="c4"><a name="h.bgliaogofoy0"></a><span>8.18</span></h1><h3 class="c4"><a name="h.8u5x2xe0ixdw"></a><span>1.Ligate 5,4</span></h3><h3 class="c4"><a name="h.8u5x2xe0ixdw"></a><span>2.transform &nbsp;pSB1C3</span></h3><h3 class="c4"><a name="h.8u5x2xe0ixdw"></a><span>8.19</span></h3><h3 class="c4"><a name="h.8u5x2xe0ixdw"></a><span>1.culture rhizobium on TY medium</span></h3><h3 class="c4"><a name="h.8u5x2xe0ixdw"></a><span>2.prepare CM,M9 medium</span></h3><h3 class="c4"><a name="h.8u5x2xe0ixdw"></a><span>3.LeuB PCR</span></h3><h3 class="c4"><a name="h.8u5x2xe0ixdw"></a><span>4. Presrvation species of Bean strain</span></h3><h3 class="c4"><a name="h.wuw8mhckhe1z"></a><span>5. Extract plasmid and Digest</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c10"><p class="c4"><span>number</span></p></td><td class="c10"><p class="c4"><span>18</span></p></td><td class="c10"><p class="c4"><span>3</span></p></td><td class="c10"><p class="c4"><span>2+6</span></p></td><td class="c10"><p class="c4"><span>16</span></p></td><td class="c10"><p class="c4"><span>11</span></p></td><td class="c16"><p class="c4"><span>14+19</span></p></td><td class="c16"><p class="c4"><span>13</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>DNA</span></p></td><td class="c10"><p class="c4"><span>12</span></p></td><td class="c10"><p class="c4"><span>10</span></p></td><td class="c10"><p class="c4"><span>18</span></p></td><td class="c10"><p class="c4"><span>9</span></p></td><td class="c10"><p class="c4"><span>7</span></p></td><td class="c16"><p class="c4"><span>7</span></p></td><td class="c16"><p class="c4"><span>6</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>Buffer2</span></p></td><td class="c10"><p class="c4"><span>5</span></p></td><td class="c10"><p class="c4"><span>5</span></p></td><td class="c10"><p class="c4"><span>5</span></p></td><td class="c10"><p class="c4"><span>5</span></p></td><td class="c10"><p class="c4"><span>5</span></p></td><td class="c16"><p class="c4"><span>5</span></p></td><td class="c16"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>BSA</span></p></td><td class="c10"><p class="c4"><span>0.5</span></p></td><td class="c10"><p class="c4"><span>0.5</span></p></td><td class="c10"><p class="c4"><span>0.5</span></p></td><td class="c10"><p class="c4"><span>0.5</span></p></td><td class="c10"><p class="c4"><span>0.5</span></p></td><td class="c16"><p class="c4"><span>0.5</span></p></td><td class="c16"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>ddH2O</span></p></td><td class="c10"><p class="c4"><span>30.1</span></p></td><td class="c10"><p class="c4"><span>31.5</span></p></td><td class="c10"><p class="c4"><span>24.1</span></p></td><td class="c10"><p class="c4"><span>33.1</span></p></td><td class="c10"><p class="c4"><span>35.1</span></p></td><td class="c16"><p class="c4"><span>35.1</span></p></td><td class="c16"><p class="c4"><span>36.1</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>enzyme</span></p></td><td class="c10"><p class="c4"><span>EcoRI1.2</span></p></td><td class="c10"><p class="c4"><span>Xba&#8544;1.2</span></p></td><td class="c10"><p class="c4"><span>EcoRI1.2</span></p></td><td class="c10"><p class="c4"><span>Xba&#8544;1.2</span></p></td><td class="c10"><p class="c4"><span>EcoRI1.2</span></p></td><td class="c16"><p class="c4"><span>EcoRI1.2</span></p></td><td class="c16"><p class="c4"><span>Xba&#8544;1.2</span></p></td></tr><tr class="c12"><td class="c10"><p class="c2"><span></span></p></td><td class="c10"><p class="c4"><span>Spe&#8544;1.2</span></p></td><td class="c10"><p class="c4"><span>Pst&#8544;1.2</span></p></td><td class="c10"><p class="c4"><span>Spe&#8544;1.2</span></p></td><td class="c10"><p class="c4"><span>Pst&#8544;1.2</span></p></td><td class="c10"><p class="c4"><span>Spe&#8544;1.2</span></p></td><td class="c16"><p class="c4"><span>Spe&#8544;1.2</span></p></td><td class="c16"><p class="c4"><span>Pst&#8544;1.2</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.79kmvmm53rae"></a><span>8.20</span></h1><h3 class="c4"><a name="h.rb7ae2jhzsxo"></a><span>1. Presrvation species of 5+4 and extract plasmid,measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span>ng/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>99.5</span></p></td><td class="c9"><p class="c4"><span>2.098</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>140.5</span></p></td><td class="c9"><p class="c4"><span>2.162</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>99</span></p></td><td class="c9"><p class="c4"><span>2.152</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>4</span></p></td><td class="c9"><p class="c4"><span>123.5</span></p></td><td class="c9"><p class="c4"><span>2.093</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>5</span></p></td><td class="c9"><p class="c4"><span>137.5</span></p></td><td class="c9"><p class="c4"><span>2.083</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.cd31d7gjoaj9"></a><span>2. Digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>number</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c6"><p class="c4"><span>4</span></p></td><td class="c6"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>DNA</span></p></td><td class="c0"><p class="c4"><span>1.3</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>1.3</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>Buffer3</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>BSA</span></p></td><td class="c0"><p class="c4"><span>0.1</span></p></td><td class="c0"><p class="c4"><span>0.1</span></p></td><td class="c0"><p class="c4"><span>0.1</span></p></td><td class="c6"><p class="c4"><span>0.1</span></p></td><td class="c6"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>ddH2O</span></p></td><td class="c0"><p class="c4"><span>7.1</span></p></td><td class="c0"><p class="c4"><span>7.4</span></p></td><td class="c0"><p class="c4"><span>7.1</span></p></td><td class="c6"><p class="c4"><span>7.4</span></p></td><td class="c6"><p class="c4"><span>7.4</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>EcoR&#8544;</span></p></td><td class="c0"><p class="c4"><span>0.25</span></p></td><td class="c0"><p class="c4"><span>0.25</span></p></td><td class="c0"><p class="c4"><span>0.25</span></p></td><td class="c6"><p class="c4"><span>0.25</span></p></td><td class="c6"><p class="c4"><span>0.25</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>Pst&#8544;</span></p></td><td class="c0"><p class="c4"><span>0.25</span></p></td><td class="c0"><p class="c4"><span>0.25</span></p></td><td class="c0"><p class="c4"><span>0.25</span></p></td><td class="c6"><p class="c4"><span>0.25</span></p></td><td class="c6"><p class="c4"><span>0.25</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c49 c4"><a name="h.wlf6j8rjal3t"></a></h1><h1 class="c4"><a name="h.urk4as897tek"></a><span>8.22</span></h1><h3 class="c4"><a name="h.dwwnj6olsp9f"></a><span>1&#12289;Presrvation species of 5+4 and extract plasmid,measure density, digest</span></h3><h3 class="c4"><a name="h.4k6v2rq1fzij"></a><span>2&#12289;pick single colonies of 19-14&amp;13&#65307;2-6&amp;3 , 11&amp;16 and culture in shaker</span></h3><h3 class="c4"><a name="h.k3thznec7nhf"></a><span>3&#12289;PCR Leu&#65288;50ul&#65289;&#65292;recover and check</span></h3><p class="c2"><span></span></p><p class="c4"><img height="197" src="images/image04.jpg" width="187"></p><p class="c4"><span>1</span><span class="c3">0&times;</span><span>Taq Buffer(with MgSO4) 5&mu;L</span></p><p class="c4"><span>MgCl2 &nbsp;4&mu;L</span></p><p class="c4"><span>Primer-F 1.5&mu;L</span></p><p class="c4"><span>Primer-R 1.5&mu;L</span></p><p class="c4"><span>H2O 33.5&mu;L</span></p><p class="c4"><span>dNTP 2&mu;L</span></p><p class="c4"><span>DNA 2&mu;L</span></p><p class="c4"><span>Taq 0.25&mu;L</span></p><p class="c4"><span>Pfu 0.25&mu;L &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span></p><p class="c4 c22"><span>Recommended By Advicer Tan</span></p><p class="c4 c22"><span>Result: success</span></p><p class="c2"><span></span></p><p class="c2"><span></span></p><p class="c2"><span></span></p><p class="c2"><span></span></p><h1 class="c49 c4"><a name="h.2yfk6buo0k2e"></a></h1><h1 class="c4"><a name="h.2yfk6buo0k2e"></a><span>8.23</span></h1><h3 class="c4"><a name="h.g493ims3jbp8"></a><span>1&#12289;Presrvation species of 2 6&amp;3&#65292;11&amp;16&#65292;14 19&amp;13 and extract plasmid, measure density&#65292;digest</span></h3><h3 class="c4"><a name="h.tknykw38j7c4"></a><span>density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c15"><p class="c4"><span>number</span></p></td><td class="c30"><p class="c4"><span>2 6&amp;3</span></p></td><td class="c54"><p class="c4"><span>11&amp;16</span></p></td><td class="c51"><p class="c4"><span>1419&amp;13</span><span class="c3">&#61569;</span></p></td><td class="c41"><p class="c4"><span class="c3">&#61570;</span></p></td><td class="c30"><p class="c4"><span class="c3">&#61571;</span></p></td><td class="c39"><p class="c4"><span class="c3">&#61572;</span></p></td><td class="c39"><p class="c4"><span class="c3">&#61573;</span></p></td></tr><tr class="c12"><td class="c15"><p class="c4"><span>density</span></p><p class="c4"><span>&#65288;ng/ul&#65289;</span></p></td><td class="c30"><p class="c4"><span>30</span></p></td><td class="c54"><p class="c4"><span>49</span></p></td><td class="c51"><p class="c4"><span>45</span></p></td><td class="c41"><p class="c4"><span>132</span></p></td><td class="c30"><p class="c4"><span>62</span></p></td><td class="c39"><p class="c4"><span>42</span></p></td><td class="c39"><p class="c4"><span>64</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.r1xl2xgbvvgi"></a><span>Digestion</span></h3><h3 class="c4"><a name="h.qvlzmnr7s30"></a><span>Single enzyme</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c30"><p class="c2"><span></span></p></td><td class="c31"><p class="c4"><span>2 6&amp;3</span></p></td><td class="c30"><p class="c4"><span>11&amp;16</span></p></td><td class="c35"><p class="c4"><span>1419&amp;13</span><span class="c3">&#61569;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61570;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61571;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61572;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61573;</span></p></td></tr><tr class="c12"><td class="c30"><p class="c4"><span>DNA</span></p></td><td class="c31"><p class="c4"><span>3.3</span></p></td><td class="c30"><p class="c4"><span>2</span></p></td><td class="c35"><p class="c4"><span>2.2</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1.5</span></p></td><td class="c8"><p class="c4"><span>2.3</span></p></td><td class="c8"><p class="c4"><span>1.5</span></p></td></tr><tr class="c12"><td class="c30"><p class="c4"><span>Buffer4</span></p></td><td class="c31"><p class="c4"><span>1</span></p></td><td class="c30"><p class="c4"><span>1</span></p></td><td class="c35"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c30"><p class="c4"><span>DdH2O</span></p></td><td class="c31"><p class="c4"><span>5.4</span></p></td><td class="c30"><p class="c4"><span>6.6</span></p></td><td class="c35"><p class="c4"><span>6.4</span></p></td><td class="c8"><p class="c4"><span>7.6</span></p></td><td class="c8"><p class="c4"><span>7.1</span></p></td><td class="c8"><p class="c4"><span>6.3</span></p></td><td class="c8"><p class="c4"><span>7.1</span></p></td></tr><tr class="c12"><td class="c30"><p class="c4"><span>XbaI</span></p></td><td class="c31"><p class="c4"><span>0.3</span></p></td><td class="c30"><p class="c4"><span>0.3</span></p></td><td class="c35"><p class="c4"><span>0.3</span></p></td><td class="c8"><p class="c4"><span>0.3</span></p></td><td class="c8"><p class="c4"><span>0.3</span></p></td><td class="c8"><p class="c4"><span>0.3</span></p></td><td class="c8"><p class="c4"><span>0.3</span></p></td></tr><tr class="c12"><td class="c30"><p class="c4"><span>BSA</span></p></td><td class="c31"><p class="c4"><span>0.1</span></p></td><td class="c30"><p class="c4"><span>0.1</span></p></td><td class="c35"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.78jdujd9b23x"></a><span>Double enzymes</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c33"><p class="c2"><span></span></p></td><td class="c8"><p class="c4"><span>2 6&amp;3</span></p></td><td class="c25"><p class="c4"><span>11&amp;16</span></p></td><td class="c35"><p class="c4"><span>1419&amp;13</span><span class="c3">&#61569;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61570;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61571;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61572;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61573;</span></p></td></tr><tr class="c12"><td class="c33"><p class="c4"><span>DNA</span></p></td><td class="c8"><p class="c4"><span>3.3</span></p></td><td class="c25"><p class="c4"><span>2</span></p></td><td class="c35"><p class="c4"><span>2.2</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1.5</span></p></td><td class="c8"><p class="c4"><span>2.3</span></p></td><td class="c8"><p class="c4"><span>1.5</span></p></td></tr><tr class="c12"><td class="c33"><p class="c4"><span>Buffer4</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c25"><p class="c4"><span>1</span></p></td><td class="c35"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c33"><p class="c4"><span>BSA</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td><td class="c25"><p class="c4"><span>0.1</span></p></td><td class="c35"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c33"><p class="c4"><span>H2O</span></p></td><td class="c8"><p class="c4"><span>5.1</span></p></td><td class="c25"><p class="c4"><span>6.4</span></p></td><td class="c35"><p class="c4"><span>6.2</span></p></td><td class="c8"><p class="c4"><span>7.4</span></p></td><td class="c8"><p class="c4"><span>6.9</span></p></td><td class="c8"><p class="c4"><span>6.1</span></p></td><td class="c8"><p class="c4"><span>6.9</span></p></td></tr><tr class="c12"><td class="c33"><p class="c4"><span>EcoRI-HF</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c25"><p class="c4"><span>0.25</span></p></td><td class="c35"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td></tr><tr class="c12"><td class="c33"><p class="c4"><span>SpeI</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c25"><p class="c4"><span>0.25</span></p></td><td class="c35"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.uosio89ahtai"></a><span>result&#65306;success</span></h3><h3 class="c4"><a name="h.oapjx65rnz4x"></a><span>2&#12289;</span><span class="c36">Preservation</span><span>&nbsp;species of CFN42 WT and BL21</span></h3><h3 class="c4"><a name="h.oapjx65rnz4x"></a><span>3&#12289;check 18&amp;3 be small gel &mdash;&mdash;succeed</span></h3><h3 class="c4"><a name="h.oapjx65rnz4x"></a><span>4&#12289;Prepare medium&#65306;LB liquid medium &nbsp;300ml&#65292;</span></h3><h3 class="c4"><a name="h.oapjx65rnz4x"></a><span>LB medium 150ml </span></h3><h3 class="c4"><a name="h.oapjx65rnz4x"></a><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;TY medium 150ml</span></h3><h3 class="c4"><a name="h.oapjx65rnz4x"></a><span>5&#12289;Prepare SMM medium&#65292;+ pro+ Vb1</span></h3><h1 class="c4"><a name="h.r6ryyomviv4b"></a><span>8.24</span></h1><h3 class="c4"><a name="h.ciewiyq5t66a"></a><span>1&#12289;Prepare SMM medium &nbsp;:MM+pro+Vb1 &nbsp;MM+pro+Vb1+leu</span></h3><h3 class="c4"><a name="h.ciewiyq5t66a"></a><span>2&#12289;check by small gel</span></h3><h1 class="c4"><a name="h.he4e0lgvyqrw"></a><span>8.25</span></h1><h3 class="c4"><a name="h.8kqf064rpjw5"></a><a href="#" name="id.b6b2514dfea5"></a><span>1&#12289;ligate 11-16&amp;18-13 and 5&amp;4 using 3A</span></h3><h3 class="c4"><a name="h.tkxpq18ksh8e"></a><span>Measure density&#65306;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>11-16</span></p></td><td class="c1"><p class="c4"><span>18-13</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>density&#65288;ng/ul&#65289;</span></p></td><td class="c1"><p class="c4"><span>46.5</span></p></td><td class="c1"><p class="c4"><span>136.5</span></p></td><td class="c1"><p class="c4"><span>85.5</span></p></td><td class="c1"><p class="c4"><span>71</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.f8muhyhuv6l2"></a><span>Double enzymes</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>18-3</span></p></td><td class="c1"><p class="c4"><span>11-16</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>DNA</span></p></td><td class="c1"><p class="c4"><span>12</span></p></td><td class="c1"><p class="c4"><span>10</span></p></td><td class="c1"><p class="c4"><span>10</span></p></td><td class="c1"><p class="c4"><span>25</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Buffer4</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>BSA</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>H2O</span></p></td><td class="c1"><p class="c4"><span>29.5</span></p></td><td class="c1"><p class="c4"><span>32.5</span></p></td><td class="c1"><p class="c4"><span>31.5</span></p></td><td class="c1"><p class="c4"><span>16.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>enzyme1</span></p></td><td class="c1"><p class="c4"><span>EcoRI-HF 1.5</span></p></td><td class="c1"><p class="c4"><span>XbaI &nbsp;1</span></p></td><td class="c1"><p class="c4"><span>EcoRI-HF &nbsp;1.5</span></p></td><td class="c1"><p class="c4"><span>XbaI &nbsp;1.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>enzyme 2</span></p></td><td class="c1"><p class="c4"><span>SpeI 1.5</span></p></td><td class="c1"><a href="#" name="id.5ffd2b59663f"></a><p class="c4"><span>PstI &nbsp;1</span></p></td><td class="c1"><p class="c4"><span>SpeI &nbsp;1.5</span></p></td><td class="c1"><p class="c4"><span>PstI &nbsp;1.5</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.ynposbrw4sji"></a><span>Check by gel</span></h3><h3 class="c4"><a name="h.sundqa7jgp4t"></a><span>Ligation of 5&amp;4</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>5</span></p></td><td class="c0"><p class="c4"><span>4</span></p></td><td class="c0"><p class="c4"><span>AC</span></p></td><td class="c0"><p class="c4"><span>T4</span></p></td><td class="c6"><p class="c4"><span>Buffer</span></p></td><td class="c6"><p class="c4"><span>H2O</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>6</span></p></td><td class="c0"><p class="c4"><span>4</span></p></td><td class="c0"><p class="c4"><span>4</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>2</span></p></td><td class="c6"><p class="c4"><span>3</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.ezohoz16dnao"></a><span>Ligation of &nbsp;11-16&amp;18-13&#65306;&#65288;ul&#65289;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>K381001</span></p></td><td class="c0"><p class="c4"><span>18-3</span></p></td><td class="c0"><p class="c4"><span>11-16</span></p></td><td class="c0"><p class="c4"><span>Buffer</span></p></td><td class="c6"><p class="c4"><span>T4</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>5</span></p></td><td class="c0"><p class="c4"><span>6</span></p></td><td class="c0"><p class="c4"><span>6</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.8ylqa0m2w31v"></a><span>2&#12289;inoculate HB101 and BL21 into the solid medium (MM+VB1+Pro and MM+VB1+Pro+Leu)&#65292;culture the Rhizobium etli CFN42 and Sinorhizobium meliloti 1021 on TY solid medium at 30&#8451;</span></h3><h3 class="c29 c4"><a name="h.94y273j4x26h"></a></h3><h1 class="c4"><a name="h.b25s7bsf4qft"></a><span>8.27</span></h1><h3 class="c4"><a name="h.94y273j4x26h"></a><span>1&#12289;transform 18-3&amp;11</span></h3><h3 class="c4"><a name="h.94y273j4x26h"></a><span>2&#12289;pick 3 colonies of 5&amp;4 and culture in l LB liquid medium</span></h3><h3 class="c4"><a name="h.94y273j4x26h"></a><span>3&#12289;culture LeuB and K115001 in LB liquid medium</span></h3><h3 class="c4"><a name="h.94y273j4x26h"></a><span>4&#12289;recover 22,21,3 and ligate 21&amp;3,22&amp;3</span></h3><h3 class="c4"><a name="h.k3ru3lgh65ye"></a><a href="#" name="id.887d11515b76"></a><span>Ligation of 21&amp;3&#65306;(ul)</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c4"><span>22</span></p></td><td class="c1"><p class="c4"><span>3</span></p></td><td class="c1"><p class="c4"><span>T4</span></p></td><td class="c1"><p class="c4"><span>Buffer</span></p></td><td class="c1"><p class="c4"><span>ddH2O</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>7</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>6</span></p></td></tr></tbody></table><h3 class="c29 c4"><a name="h.6pt8yxfqhwg6"></a></h3><h3 class="c4"><a name="h.6pt8yxfqhwg6"></a><span>Ligation of &nbsp;22&amp;3&#65306;(ul)</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c4"><span>21</span></p></td><td class="c1"><p class="c4"><span>3</span></p></td><td class="c1"><p class="c4"><span>T4</span></p></td><td class="c1"><p class="c4"><span>Buffer</span></p></td><td class="c1"><p class="c4"><span>ddH2O</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>8.5</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>4.5</span></p></td></tr></tbody></table><p class="c2"><span></span></p><p class="c2"><span></span></p><h1 class="c4"><a name="h.5bliw8b6jufq"></a><span>8.28</span></h1><h3 class="c4"><a name="h.ee824wmlivl1"></a><span>1&#12289;Prepare medium&#65306;LB liquid medium &nbsp;500ml</span></h3><h3 class="c4"><a name="h.ee824wmlivl1"></a><span>2&#12289;transform 5&amp;4 and 18-3&amp;11-16 again&#65292;and transform leu 21&amp;3 and 22&amp;3</span></h3><h3 class="c4"><a name="h.ee824wmlivl1"></a><span>3&#12289; culture deficient cells</span></h3><h3 class="c4"><a name="h.ee824wmlivl1"></a><span>4&#12289;culture 18-3&amp;11 in LB liquid medium </span></h3><h3 class="c4"><a name="h.ee824wmlivl1"></a><span>8.29</span></h3><h3 class="c4"><a name="h.ee824wmlivl1"></a><span>1&#12289;PCR :5&amp;4 and leu</span></h3><h3 class="c4"><a name="h.awlrj81666pu"></a><span>PCR&#65288;ul&#65289;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c17"><p class="c4"><span>ddH2O</span></p></td><td class="c17"><p class="c4"><span>dNTP</span></p></td><td class="c21"><p class="c4"><span>Buffer</span></p></td><td class="c17"><a href="#" name="id.bd219e189f6f"></a><p class="c4"><span>Primer-F</span></p></td><td class="c21"><p class="c4"><span>Primer-R</span></p></td><td class="c21"><p class="c4"><span>dna</span></p></td><td class="c21"><p class="c4"><span>Taq</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>39.5</span></p></td><td class="c17"><p class="c4"><span>2</span></p></td><td class="c21"><p class="c4"><span>5</span></p></td><td class="c17"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.cbhr3fgm4nrj"></a><span>2&#12289;Presrvation species of 18-3&amp;11 and extract plasmid,measure density, digest</span></h3><h3 class="c4"><a name="h.cbhr3fgm4nrj"></a><span>density&#65288;1&#65289;19.5&#65288;2&#65289;130.5</span></h3><h3 class="c4"><a name="h.cbhr3fgm4nrj"></a><span>digestion</span></h3><h3 class="c4"><a name="h.p91mu85t6yz4"></a><span>single enzyme</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>&#65288;1&#65289;</span></p></td><td class="c9"><p class="c4"><span>&#65288;2&#65289;</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>6</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>BSA</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>2.65</span></p></td><td class="c9"><p class="c4"><span>7.65</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>XbaI</span></p></td><td class="c9"><p class="c4"><span>0.35</span></p></td><td class="c9"><p class="c4"><span>0.35</span></p></td></tr></tbody></table><p class="c4"><span>Double enzymes:</span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>&#65288;1&#65289;</span></p></td><td class="c9"><p class="c4"><span>&#65288;2&#65289;</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>6</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>BSA</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>2.4</span></p></td><td class="c9"><p class="c4"><span>7.4</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>EcoRI</span></p></td><td class="c9"><p class="c4"><span>0.25</span></p></td><td class="c9"><p class="c4"><span>0.25</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>PstI</span></p></td><td class="c9"><p class="c4"><span>0.25</span></p></td><td class="c9"><p class="c4"><span>0.25</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.71fxa3curofb"></a><span>Check by small gel</span></h3><h3 class="c4"><a name="h.71fxa3curofb"></a><span>3&#12289;pick single colony of 5&amp;4&#65292;leu,21&amp;3,22&amp;3&#65292;18-3&amp;11-16 and culture in LB liquid medium&#12290;</span></h3><h3 class="c4"><a name="h.71fxa3curofb"></a><span>4&#12289;prepare TY medium 100ml</span></h3><h3 class="c4"><a name="h.71fxa3curofb"></a><span>8.30</span></h3><h3 class="c4"><a name="h.71fxa3curofb"></a><a href="#" name="id.e846d4e866ce"></a><span>1&#12289;Presrvation species of 5&amp;4,leuB89,leuB13 and extract plasmid,measure density, digest</span></h3><h3 class="c4"><a name="h.71fxa3curofb"></a><span>density&#65306;</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c17"><p class="c4"><span>leuB13&#65288;1&#65289;</span></p></td><td class="c17"><a href="#" name="id.f54c4c23a77f"></a><p class="c4"><span>5&amp;4&#65288;1&#65289;</span></p></td><td class="c21"><p class="c4"><span>5&amp;4&#65288;2&#65289;</span></p></td><td class="c17"><p class="c4"><span>5&amp;4&#65288;3&#65289;</span></p></td><td class="c21"><p class="c4"><span>leuB89&#65288;1&#65289;</span></p></td><td class="c21"><p class="c4"><span>leuB89&#65288;2&#65289;</span></p></td><td class="c21"><p class="c4"><span>leuB89(3)</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>24.5</span></p></td><td class="c17"><p class="c4"><span>188.0</span></p></td><td class="c21"><p class="c4"><span>62.0</span></p></td><td class="c17"><p class="c4"><span>132.0</span></p></td><td class="c21"><p class="c4"><span>6.8</span></p></td><td class="c21"><p class="c4"><span>3.1</span></p></td><td class="c21"><p class="c4"><span>10.8</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.6jtrl26b1tlv"></a><span>Digestion of &nbsp;5&amp;4</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>&#65288;1&#65289;</span></p></td><td class="c13"><p class="c4"><span>&#65288;2&#65289;</span></p></td><td class="c13"><p class="c4"><span>&#65288;3&#65289;</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DNA</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer3</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>ddH2O</span></p></td><td class="c5"><p class="c4"><span>7.5</span></p></td><td class="c13"><p class="c4"><span>6.5</span></p></td><td class="c13"><p class="c4"><span>7.5</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>BSA</span></p></td><td class="c5"><p class="c4"><span>0.1</span></p></td><td class="c13"><p class="c4"><span>0.1</span></p></td><td class="c13"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>EcoRI</span></p></td><td class="c5"><p class="c4"><span>0.2</span></p></td><td class="c13"><p class="c4"><span>0.2</span></p></td><td class="c13"><p class="c4"><span>0.2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>PstI</span></p></td><td class="c5"><p class="c4"><span>0.2</span></p></td><td class="c13"><p class="c4"><span>0.2</span></p></td><td class="c13"><p class="c4"><span>0.2</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.9sbx477gdv2a"></a><span>Check by small gel</span></h3><h3 class="c4"><a name="h.9sbx477gdv2a"></a><span>Result:5&amp;4 and leuB successfully ligate</span></h3><h1 class="c4"><a name="h.em1ctnutxs3l"></a><span>8.31</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.3gv9af27ikw"></a><span>1&#12289;did enzyme digestion to 2 part (with psB1AK2 carrier) and K115001 part(with psB1AT3 carrier) and run electrophoresis together with psB1C3. We recycled the carrier part by cutting the gel.</span></h3><h3 class="c4"><a name="h.3gv9af27ikw"></a><span>density&#65306;</span></h3><h3 class="c4"><a name="h.3gv9af27ikw"></a><span>2 part &nbsp; 37.5</span></h3><a href="#" name="id.a5fc2f8264e3"></a><h3 class="c4"><a name="h.3gv9af27ikw"></a><span>K115001 part &nbsp; 120</span></h3><h3 class="c4"><a name="h.p7f0i2i3brxd"></a><span>digestion(ul)</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>K115001</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>9</span></p></td><td class="c9"><p class="c4"><span>9</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>10*H Buffer</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>EcoRI</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>PstI</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>7</span></p></td><td class="c9"><p class="c4"><span>7</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.ox56qgmq045h"></a><span>9.1</span></h1><h3 class="c4"><a name="h.q4spboi6k88h"></a><span>1&#12289;prepare 0147TY medium</span></h3><h3 class="c4"><a name="h.q4spboi6k88h"></a><span>Tryptone &nbsp;5.0g &nbsp;Yeast extract 3.0g &nbsp;CaCl.6H2O &nbsp;1.3g &nbsp;Agar &nbsp;15.0g Distilled water 1.0L</span></h3><h3 class="c4"><a name="h.q4spboi6k88h"></a><span>2&#12289;ligate 18-3&amp;11-16 and 18-3&amp;11</span></h3><h3 class="c4"><a name="h.q4spboi6k88h"></a><span>density&#65306;</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c10"><p class="c4"><span>18-3&#65288;1&#65289;</span></p></td><td class="c10"><p class="c4"><span>18-3&#65288;2&#65289;</span></p></td><td class="c10"><p class="c4"><span>11-16&#65288;1&#65289;</span></p></td><td class="c10"><p class="c4"><span>11-16&#65288;2&#65289;</span></p></td><td class="c10"><p class="c4"><span>K carrier&#65288;1&#65289;</span></p></td><td class="c10"><p class="c4"><span>K carrier&#65288;2&#65289;</span></p></td><td class="c16"><p class="c4"><span>AC&#65288;1&#65289;</span></p></td><td class="c16"><p class="c4"><span>AC(2)</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>140</span></p></td><td class="c10"><p class="c4"><span>98</span></p></td><td class="c10"><p class="c4"><span>278.5</span></p></td><td class="c10"><p class="c4"><span>203&#65294;0</span></p></td><td class="c10"><p class="c4"><span>17.5</span></p></td><td class="c10"><p class="c4"><span>17.5</span></p></td><td class="c16"><p class="c4"><span>7.9</span></p></td><td class="c16"><p class="c4"><span>13.1</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.hedvpls048g0"></a><span>Digestion:<br></span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>18-3</span></p></td><td class="c13"><p class="c4"><span>11</span></p></td><td class="c13"><p class="c4"><span>11-16</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DNA</span></p></td><td class="c5"><p class="c4"><span>7</span></p></td><td class="c13"><p class="c4"><span>10</span></p></td><td class="c13"><p class="c4"><span>4</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer</span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>enzyme1</span></p></td><td class="c5"><p class="c4"><span>E &nbsp;1</span></p></td><td class="c13"><p class="c4"><span>X &nbsp; 1</span></p></td><td class="c13"><p class="c4"><span>X &nbsp;1</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>enzyme 2</span></p></td><td class="c5"><p class="c4"><span>S &nbsp;1</span></p></td><td class="c13"><p class="c4"><span>P &nbsp; &nbsp;1</span></p></td><td class="c13"><p class="c4"><span>P &nbsp; 1</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DdH2O</span></p></td><td class="c5"><p class="c4"><span>9</span></p></td><td class="c13"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>12</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.3yfownuunxbz"></a><span>Ligation:</span></h3><h3 class="c4"><a name="h.3yfownuunxbz"></a><span>18-3&#65306;3ul &nbsp;16:1ul &nbsp; 2&#65306;6ul &nbsp;T4&#65306;1ul &nbsp;Buffer&#65306;2ul &nbsp; H2O&#65306;7ul</span></h3><h3 class="c29 c4"><a name="h.3yfownuunxbz"></a></h3><h3 class="c4"><a name="h.3yfownuunxbz"></a><span>18-3&#65306;3ul &nbsp;11-16&#65306;4ul &nbsp;2&#65306;6ul &nbsp; T4&#65306;1ul &nbsp; Buffer&#65306;2ul &nbsp;ddH2O&#65306;4ul</span></h3><h1 class="c4"><a name="h.tmngci922iwu"></a><span>9.2 </span></h1><h3 class="c4"><a name="h.wkppfbc5lyza"></a><span>1&#12289;Presrvation species of leuB and extract plasmid,measure density, digest</span></h3><h3 class="c4"><a name="h.wkppfbc5lyza"></a><span>density&#65306;</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><h3 class="c4"><a name="h.wkppfbc5lyza"></a><span>1</span></h3></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>3</span></p></td><td class="c13"><p class="c4"><span>4</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>292.5</span></p></td><td class="c5"><p class="c4"><span>21.5</span></p></td><td class="c13"><p class="c4"><span>223.5</span></p></td><td class="c13"><p class="c4"><span>32.0</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.bi0spzoaltti"></a><span>Digestion:</span></h1><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>leuB</span></p></td><td class="c9"><a href="#" name="id.d705116cf752"></a><p class="c4"><span>K(pSB1C3)</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>EcoRI</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>PstI</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>14</span></p></td><td class="c9"><p class="c4"><span>8.5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>7.5</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.9sfoto1zfgis"></a><span>Check by small gel:</span></h3><p class="c2"><span></span></p><h3 class="c4"><a name="h.bjhs1az2y57t"></a><span>recover&#65306;</span></h3><h3 class="c4"><a name="h.bjhs1az2y57t"></a><span>2&#12289;pick single colony of 18-3&amp;11-16 and 18-3&amp;11 and culture in shaker</span></h3><h3 class="c4"><a name="h.bjhs1az2y57t"></a><span>3&#12289;transform BBa_I751250,as LuxI_AHL producer&#12290;Transform BBa_J04450(placI+mRFP)</span></h3><h3 class="c4"><a name="h.bjhs1az2y57t"></a><span>9.3</span></h3><h3 class="c4"><a name="h.bjhs1az2y57t"></a><span>1&#12289;ligate leuB, K(pSB1C3) carrier and transform</span></h3><h3 class="c4"><a name="h.bjhs1az2y57t"></a><span>density&#65306;</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>Before Concentration</span></p></td><td class="c9"><p class="c4"><span>After &nbsp;Concentration</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>LeuB</span></p></td><td class="c9"><p class="c4"><span>15</span></p></td><td class="c9"><p class="c4"><span>36</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1C3</span></p></td><td class="c9"><p class="c4"><span>8</span></p></td><td class="c9"><p class="c4"><span>25</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.6ii9ncjvqxui"></a><span>ligation</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c4"><span>LeuB</span></p></td><td class="c1"><p class="c4"><span>Psb1c3</span></p></td><td class="c1"><p class="c4"><span>T4</span></p></td><td class="c1"><p class="c4"><span>Buffer</span></p></td><td class="c1"><p class="c4"><span>DdH2O</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>7</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.im43403pxmes"></a><span>2&#12289;extract plasmid of 8-3&amp;11-16 and 18-3&amp;11, digest&#65292;and check by small gel</span></h3><h3 class="c4"><a name="h.im43403pxmes"></a><span>Density:</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c34"><p class="c4"><span>18-3&amp;11-161</span></p></td><td class="c34"><p class="c4"><span>18-3&amp;11-162</span></p></td><td class="c18"><p class="c4"><span>18-3&amp;11 1</span></p></td><td class="c18"><a href="#" name="id.ad39c9b90daa"></a><p class="c4"><span>18-3&amp;11 2</span></p></td><td class="c52"><p class="c4"><span>5-4&#65288;AC&#65289;1 </span></p></td><td class="c56"><p class="c4"><span>5-4&#65288;AC&#65289; 2</span></p></td><td class="c53"><p class="c4"><span>2-6-3</span></p></td></tr><tr class="c12"><td class="c34"><p class="c4"><span>265</span></p></td><td class="c34"><p class="c4"><span>242</span></p></td><td class="c18"><p class="c4"><span>192</span></p></td><td class="c18"><p class="c4"><span>217.5</span></p></td><td class="c52"><p class="c4"><span>193</span></p></td><td class="c56"><p class="c4"><span>72</span></p></td><td class="c53"><p class="c4"><span>63.5</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.qevj1yc7kkwj"></a><span>digestion&#65306;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>18-3&amp;11-16(1)</span></p></td><td class="c1"><p class="c4"><span>18-3&amp;11-16(2)</span></p></td><td class="c1"><p class="c4"><span>18-3&amp;11(1)</span></p></td><td class="c1"><p class="c4"><span>18-3&amp;11(2)</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>DNA</span></p></td><td class="c1"><p class="c4"><span>3.5</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>4.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Buffer</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>EcoRI</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>PstI</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>ddH2O</span></p></td><td class="c1"><p class="c4"><span>12.5</span></p></td><td class="c1"><p class="c4"><span>12</span></p></td><td class="c1"><p class="c4"><span>11</span></p></td><td class="c1"><p class="c4"><span>11.5</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.s7dxsac1fmjh"></a><span>Check by gel&#65306;</span></h3><p class="c2"><span></span></p><h3 class="c4"><a name="h.f5jqe09y4j66"></a><span>2&#12289;ligate 5-4&amp;2-6-3</span></h3><h3 class="c4"><a name="h.6o1u60ekkzdx"></a><span>ligation</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>K</span></p></td><td class="c0"><p class="c4"><span>5-4</span></p></td><td class="c0"><p class="c4"><span>2-6-3</span></p></td><td class="c0"><p class="c4"><span>T4&#36830;&#25509;&#37238;</span></p></td><td class="c6"><p class="c4"><span>Buffer</span></p></td><td class="c6"><p class="c4"><span>DdH2O</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>6</span></p></td><td class="c0"><p class="c4"><span>4</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>2</span></p></td><td class="c6"><p class="c4"><span>5</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.h3yoy287vb0g"></a><span>9&#65294;4</span></h1><h3 class="c4"><a name="h.djojukljacfp"></a><span>1&#12289;transform 5-4&amp;2-6-3&#65292;extract plasmid of &nbsp;2-6-3&#19982;2</span></h3><h3 class="c4"><a name="h.djojukljacfp"></a><span>2&#12289;transform Pea Rhizobium</span></h3><h3 class="c4"><a name="h.djojukljacfp"></a><span>9.5</span></h3><h3 class="c4"><a name="h.djojukljacfp"></a><span>1&#12289;Presrvation species of leuB and extract plasmid,measure density, digest, PCR ,check by gel</span></h3><h3 class="c4"><a name="h.djojukljacfp"></a><span>PCR</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>2</span></p></td><td class="c6"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>ddH2O</span></p></td><td class="c0"><p class="c4"><span>37.5</span></p></td><td class="c0"><p class="c4"><span>37</span></p></td><td class="c0"><p class="c4"><span>36.5</span></p></td><td class="c6"><p class="c4"><span>36.5</span></p></td><td class="c6"><p class="c4"><span>36.5</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>dNTPmix</span></p></td><td class="c0"><p class="c4"><span>4</span></p></td><td class="c0"><p class="c4"><span>4</span></p></td><td class="c0"><p class="c4"><span>4</span></p></td><td class="c6"><p class="c4"><span>4</span></p></td><td class="c6"><p class="c4"><span>4</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>Buffer</span></p></td><td class="c0"><p class="c4"><span>5</span></p></td><td class="c0"><p class="c4"><span>5</span></p></td><td class="c0"><p class="c4"><span>5</span></p></td><td class="c6"><p class="c4"><span>5</span></p></td><td class="c6"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>VF2</span></p></td><td class="c0"><p class="c4"><span>1.5</span></p></td><td class="c0"><p class="c4"><span>1.5</span></p></td><td class="c0"><p class="c4"><span>1.5</span></p></td><td class="c6"><p class="c4"><span>1.5</span></p></td><td class="c6"><p class="c4"><span>1.5</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>VR</span></p></td><td class="c0"><p class="c4"><span>1.5</span></p></td><td class="c0"><p class="c4"><span>1.5</span></p></td><td class="c0"><p class="c4"><span>1.5</span></p></td><td class="c6"><p class="c4"><span>1.5</span></p></td><td class="c6"><p class="c4"><span>1.5</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>tamplate</span></p></td><td class="c0"><p class="c4"><span>0</span></p></td><td class="c0"><p class="c4"><span>0.5</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>Taq</span></p></td><td class="c0"><p class="c4"><span>0.5</span></p></td><td class="c0"><p class="c4"><span>0.5</span></p></td><td class="c0"><p class="c4"><span>0.5</span></p></td><td class="c6"><p class="c4"><span>0.5</span></p></td><td class="c6"><p class="c4"><span>0.5</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.4vezb87sj3gs"></a><span>Check by gel </span></h3><p class="c4"><img height="527" src="images/image02.jpg" width="527"></p><h3 class="c4"><a name="h.72c4zg6pthgu"></a><span>digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>LeuB &nbsp;1</span></p></td><td class="c9"><p class="c4"><span>LeuB &nbsp;3</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>BufferM</span></p></td><td class="c9"><p class="c4"><span>0.5</span></p></td><td class="c9"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>1.1</span></p></td><td class="c9"><p class="c4"><span>1.1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>SpeI</span></p></td><td class="c9"><p class="c4"><span>0.2</span></p></td><td class="c9"><p class="c4"><span>0.2</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>XbaI</span></p></td><td class="c9"><p class="c4"><span>0.2</span></p></td><td class="c9"><p class="c4"><span>0.2</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.prk8j67a3erf"></a><span>Check by gel </span></h3><h1 class="c4"><a name="h.ftq4x9fezl9p"></a><span>9.6</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.tzu0yabpanct"></a><span>1&#12289;Presrvation species of 54263 and extract plasmid, digest, PCR ,check by gel</span></h3><h3 class="c29 c4"><a name="h.tzu0yabpanct"></a></h3><h3 class="c4"><a name="h.3i6aolmrnp3z"></a><span>digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DNA</span></p></td><td class="c5"><p class="c4"><span>1.4</span></p></td><td class="c13"><p class="c4"><span>1.4</span></p></td><td class="c13"><p class="c4"><span>1.7</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer</span></p></td><td class="c5"><p class="c4"><span>0.5</span></p></td><td class="c13"><p class="c4"><span>0.5</span></p></td><td class="c13"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DdH2O</span></p></td><td class="c5"><p class="c4"><span>2.5</span></p></td><td class="c13"><p class="c4"><span>2.5</span></p></td><td class="c13"><p class="c4"><span>2.2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>EcoRI</span></p></td><td class="c5"><p class="c4"><span>0.3</span></p></td><td class="c13"><p class="c4"><span>0.3</span></p></td><td class="c13"><p class="c4"><span>0.3</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>PstI</span></p></td><td class="c5"><p class="c4"><span>0.3</span></p></td><td class="c13"><p class="c4"><span>0.3</span></p></td><td class="c13"><p class="c4"><span>0.3</span></p></td></tr></tbody></table><h1 class="c49 c4"><a name="h.woe6zwxsksvp"></a></h1><h1 class="c4"><a name="h.7wkxrcs7xpbv"></a><span>9.7</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.uxiz6xa9xofa"></a><span>1&#12289;measure OD of Deficient cell</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c4"><span>MM&#65288;leuB-&#65289;</span></p></td><td class="c0"><p class="c4"><span>1 &nbsp;leuB+</span></p></td><td class="c0"><p class="c4"><span>2 &nbsp; leuB+</span></p></td><td class="c6"><p class="c4"><span>3 &nbsp; leuB+</span></p></td><td class="c6"><p class="c4"><span>4 &nbsp;leuB+</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>OD&#65288;A340&#65289;</span></p></td><td class="c0"><p class="c4"><span>1.611-1</span></p></td><td class="c0"><p class="c4"><span>1.698-1</span></p></td><td class="c0"><p class="c4"><span>1.818-1</span></p></td><td class="c6"><p class="c4"><span>1.548-1</span></p></td><td class="c6"><p class="c4"><span>1.881-1</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.w97sjfemswol"></a><span>2&#12289;digest &nbsp;leuB&#12289;&#9314;&#12289;K115001</span></h3><h3 class="c4"><a name="h.mfs7a181f07r"></a><span>digestion&#65306;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c26"><p class="c2"><span></span></p></td><td class="c30"><p class="c4"><span>8</span></p></td><td class="c25"><p class="c4"><span>6</span></p></td><td class="c46"><p class="c4"><span>LeuB</span></p></td><td class="c30"><p class="c4"><span>3</span></p></td><td class="c24"><p class="c4"><span>T1</span></p></td><td class="c26"><p class="c4"><span>T2</span></p></td><td class="c24"><p class="c4"><span>T3</span></p></td></tr><tr class="c12"><td class="c26"><p class="c4"><span>DNA</span></p></td><td class="c30"><p class="c4"><span>9</span></p></td><td class="c25"><p class="c4"><span>9</span></p></td><td class="c46"><p class="c4"><span>10</span></p></td><td class="c30"><p class="c4"><span>10</span></p></td><td class="c24"><p class="c4"><span>10</span></p></td><td class="c26"><p class="c4"><span>13</span></p></td><td class="c24"><p class="c4"><span>12</span></p></td></tr><tr class="c12"><td class="c26"><p class="c4"><span>Buffer</span></p></td><td class="c30"><p class="c4"><span>2</span></p></td><td class="c25"><p class="c4"><span>2</span></p></td><td class="c46"><p class="c4"><span>2</span></p></td><td class="c30"><p class="c4"><span>2</span></p></td><td class="c24"><p class="c4"><span>2</span></p></td><td class="c26"><p class="c4"><span>2</span></p></td><td class="c24"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c26"><p class="c4"><span>DdH2O</span></p></td><td class="c30"><p class="c4"><span>7</span></p></td><td class="c25"><p class="c4"><span>6.6</span></p></td><td class="c46"><p class="c4"><span>5.6</span></p></td><td class="c30"><p class="c4"><span>5.6</span></p></td><td class="c24"><p class="c4"><span>5.6</span></p></td><td class="c26"><p class="c4"><span>2.6</span></p></td><td class="c24"><p class="c4"><span>3.6</span></p></td></tr><tr class="c12"><td class="c26"><p class="c4"><span>EcoRI</span></p></td><td class="c30"><p class="c4"><span>1</span></p></td><td class="c25"><p class="c4"><span>1.2</span></p></td><td class="c46"><p class="c4"><span>1.2</span></p></td><td class="c30"><p class="c4"><span>1.2</span></p></td><td class="c24"><p class="c4"><span>1.2</span></p></td><td class="c26"><p class="c4"><span>1.2</span></p></td><td class="c24"><p class="c4"><span>1.2</span></p></td></tr><tr class="c12"><td class="c26"><p class="c4"><span>SpeI</span></p></td><td class="c30"><p class="c4"><span>1</span></p></td><td class="c25"><p class="c4"><span>1.2</span></p></td><td class="c46"><p class="c4"><span>1.2</span></p></td><td class="c30"><p class="c4"><span>1.2</span></p></td><td class="c24"><p class="c4"><span>1.2</span></p></td><td class="c26"><p class="c4"><span>1.2</span></p></td><td class="c24"><p class="c4"><span>1.2</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.5mlvofbvkc4i"></a><span>density&#65306;</span></h3><h3 class="c4"><a name="h.5mlvofbvkc4i"></a><span>LeuB 98.0</span></h3><h3 class="c4"><a name="h.5mlvofbvkc4i"></a><span>T &nbsp;1:93.5 &nbsp; &nbsp; 2:73.5 &nbsp; &nbsp;3:87</span></h3><h3 class="c4"><a name="h.5mlvofbvkc4i"></a><span>3 &nbsp;1:97 &nbsp; &nbsp; &nbsp; 2:73 &nbsp; &nbsp; 3:82</span></h3><h3 class="c4"><a name="h.5mlvofbvkc4i"></a><span>9.8</span></h3><h3 class="c4"><a name="h.5mlvofbvkc4i"></a><span>1&#12289;extract plasmid of 6&#65292;and digest with LeuB</span></h3><h3 class="c4"><a name="h.9dqcvm6xpq2y"></a><span>Density of 6&#65306;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>57</span></p></td><td class="c9"><p class="c4"><span>68</span></p></td><td class="c9"><p class="c4"><span>50</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.ufa9zdoc4n5v"></a><span>digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>LeuB</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DNA</span></p></td><td class="c5"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>10</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer</span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DdH2O</span></p></td><td class="c5"><p class="c4"><span>9.6</span></p></td><td class="c13"><p class="c4"><span>9.6</span></p></td><td class="c13"><p class="c4"><span>5.6</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>EcoRI</span></p></td><td class="c5"><p class="c4"><span>1.2</span></p></td><td class="c13"><p class="c4"><span>1.2</span></p></td><td class="c13"><p class="c4"><span>1.2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>SpeI</span></p></td><td class="c5"><p class="c4"><span>1.2</span></p></td><td class="c13"><p class="c4"><span>1.2</span></p></td><td class="c13"><p class="c4"><span>1.2</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.tgspjxa7cy05"></a><span>2&#12289;ligate leuB&amp;3</span></h3><h3 class="c4"><a name="h.tgspjxa7cy05"></a><span>ligation&#65306;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>T4</span></p></td><td class="c0"><p class="c4"><span>Buffer</span></p></td><td class="c0"><p class="c4"><span>leuB</span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c6"><p class="c4"><span>T carrier</span></p></td><td class="c6"><p class="c4"><span>DdH2O</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c0"><p class="c4"><span>0.7</span></p></td><td class="c6"><p class="c4"><span>10</span></p></td><td class="c6"><p class="c4"><span>3.3</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.2tn7lcrp7780"></a><span>9.9</span></h1><h3 class="c4"><a name="h.ix5rjouwfyue"></a><span>1&#12289;check 6, leuB, 8</span></h3><h3 class="c4"><a name="h.ix5rjouwfyue"></a><span>Gel&#65306;</span></h3><h3 class="c4"><a name="h.ix5rjouwfyue"></a><span>Ligate 8&amp;6&#12289;6&amp;leuB</span></h3><h3 class="c4"><a name="h.jizjvyn0z6vd"></a><span>Ligation of 8&amp;6&#65306;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>T carrier</span></p></td><td class="c0"><p class="c4"><span>8</span></p></td><td class="c0"><p class="c4"><span>6</span></p></td><td class="c0"><p class="c4"><span>T4</span></p></td><td class="c6"><p class="c4"><span>Buffer</span></p></td><td class="c6"><p class="c4"><span>DdH2O</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>10</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>2</span></p></td><td class="c6"><p class="c4"><span>5</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.sojo5w7w5q9c"></a><span>Ligation of &nbsp;6&amp;leuB</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>T carrier</span></p></td><td class="c0"><p class="c4"><span>6</span></p></td><td class="c0"><p class="c4"><span>LeuB</span></p></td><td class="c0"><p class="c4"><span>T4</span></p></td><td class="c6"><p class="c4"><span>Buffer</span></p></td><td class="c6"><p class="c4"><span>DdH2O</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>10</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>2</span></p></td><td class="c6"><p class="c4"><span>3</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.vnbfx386r4a2"></a><span>9.10</span></h1><h3 class="c4"><a name="h.90ycqjb04vb7"></a><span>1&#12289;transform 8&amp;6 and &nbsp;leuB</span></h3><h3 class="c4"><a name="h.90ycqjb04vb7"></a><span>2&#12289;culture leuB&amp;3 in LB medium</span></h3><h3 class="c4"><a name="h.90ycqjb04vb7"></a><span>3&#12289;device checking&#65306;</span></h3><h3 class="c4"><a name="h.90ycqjb04vb7"></a><span>Device1 has strong Fluorescent and device 2 hasslight one</span></h3><h3 class="c4"><a name="h.90ycqjb04vb7"></a><span>Picture:</span></h3><h1 class="c4 c49"><a name="h.1ummjyzat1wc"></a></h1><h1 class="c4"><a name="h.p1bniw9xi6lt"></a><span>9.11</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.j1mlghz0dpg9"></a><span>1&#12289;PCR&#65306;sinR&#12289;pro-RBS-sinR&#12289;raiR&#12289;raiR-pro</span></h3><p class="c2"><span></span></p><h3 class="c4"><a name="h.fcdrc1dlv06o"></a><span>density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c37"><p class="c4"><span>sinI</span></p></td><td class="c37"><p class="c4"><span>12.8</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>SinI pro</span></p></td><td class="c37"><p class="c4"><span>9.6</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>Pro-RBS-sinI</span></p></td><td class="c37"><p class="c4"><span>15.1</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>RaiI</span></p></td><td class="c37"><p class="c4"><span>14.2</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>RaiI pro</span></p></td><td class="c37"><p class="c4"><span>9.4</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>Pro-RBS-raiI</span></p></td><td class="c37"><p class="c4"><span>11.4</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>SinR pro</span></p></td><td class="c37"><p class="c4"><span>11.5</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>Pro-RBS-raiR</span></p></td><td class="c37"><p class="c4"><span>15.0</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.ko2zysom0iot"></a><span>2&#12289;pick single colony of 6&amp;leuB</span></h3><h3 class="c4"><a name="h.ko2zysom0iot"></a><span>9.12</span></h3><h3 class="c4"><a name="h.ko2zysom0iot"></a><span>1&#12289;digest 21&#12289;22 and ligate</span></h3><h3 class="c4"><a name="h.ko2zysom0iot"></a><span>digestion</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>21</span></p></td><td class="c13"><p class="c4"><span>22</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer</span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>NDA</span></p></td><td class="c5"><p class="c4"><span>13.6</span></p></td><td class="c13"><p class="c4"><span>15</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DdH2O</span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>0.6</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>EcoRI</span></p></td><td class="c5"><p class="c4"><span>1.2</span></p></td><td class="c13"><p class="c4"><span>1.2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>SpeI</span></p></td><td class="c5"><p class="c4"><span>1.2</span></p></td><td class="c13"><p class="c4"><span>1.2</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.7q3di5col2wi"></a><span>ligation</span></h3><h3 class="c4"><a name="h.7q3di5col2wi"></a><span>21&amp;3</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>21</span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c0"><p class="c4"><span>Buffer </span></p></td><td class="c0"><p class="c4"><span>T4</span></p></td><td class="c6"><p class="c4"><span>DdH2O</span></p></td><td class="c6"><p class="c4"><span>T</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>3</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>3</span></p></td><td class="c6"><p class="c4"><span>10</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.8g2gy677wg7m"></a><span>22&amp;3</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>22</span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c0"><p class="c4"><span>Buffer</span></p></td><td class="c0"><p class="c4"><span>T4</span></p></td><td class="c6"><p class="c4"><span>DdH2O</span></p></td><td class="c6"><p class="c4"><span>T</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>5</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>10</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.78fosknlspxo"></a><span>2&#12289;extract plasmid of leuB-6 and measure density</span></h3><h3 class="c4"><a name="h.78fosknlspxo"></a><span>density&#65306;16.5</span></h3><h1 class="c4"><a name="h.z2gmdjiy1hon"></a><span>9&#65294;13</span></h1><h3 class="c4"><a name="h.slxf8ucbmfw5"></a><span>1&#12289;transform and culture 21&amp;3,22&amp;3,8&amp;6&#65292;recover PSB1C3 carrier</span></h3><h3 class="c4"><a name="h.xnxdtzar9e2f"></a><span>2.Pcr</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c17"><p class="c4"><span>ddH2O</span></p></td><td class="c17"><p class="c4"><span>Pfu with MgSO4 buffer</span></p></td><td class="c21"><p class="c4"><span>dNTP</span></p></td><td class="c17"><p class="c4"><span>PF</span></p></td><td class="c21"><p class="c4"><span>PR</span></p></td><td class="c21"><p class="c4"><span>Template</span></p></td><td class="c21"><p class="c4"><span>Pfu</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>38</span><span class="c3">&micro;</span><span>l</span></p></td><td class="c17"><p class="c4"><span>5</span><span class="c3">&micro;</span><span>l</span></p></td><td class="c21"><p class="c4"><span>4</span><span class="c3">&micro;</span><span>l</span></p></td><td class="c17"><p class="c4"><span>1</span><span class="c3">&micro;</span><span>l</span></p></td><td class="c21"><p class="c4"><span>1</span><span class="c3">&micro;</span><span>l</span></p></td><td class="c21"><p class="c4"><span>0.5</span><span class="c3">&micro;</span><span>l</span></p></td><td class="c21"><p class="c4"><span>0.5</span><span class="c3">&micro;</span><span>l</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.vyykrtrhy0we"></a><span>Results&#65306;failed</span></h3><h3 class="c4"><a name="h.vyykrtrhy0we"></a><span>3.digest sinR, sinI,pro- sinI</span></h3><h3 class="c4"><a name="h.2kxssry4fo1d"></a><span>Digestion:20</span><span class="c3">&micro;</span><span>l</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>sinR</span></p></td><td class="c13"><p class="c4"><span>sinI</span></p></td><td class="c13"><p class="c4"><span>pro- sinI</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DNA</span></p></td><td class="c5"><p class="c4"><span>10</span></p></td><td class="c13"><p class="c4"><span>11</span></p></td><td class="c13"><p class="c4"><span>16</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer 10</span><span class="c3">&times;</span><span>H</span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>ddH2O</span></p></td><td class="c5"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>5</span></p></td><td class="c13"><p class="c4"><span>0</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>EcoRI</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>PstI</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.3n89uor9jv7a"></a><span>9.15</span></h1><h3 class="c4"><a name="h.mwd71ak11zm"></a><span>Digestion of Leu&amp;3 :20</span><span class="c3">&micro;</span><span>l</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c4"><span>number</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DNA(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>10</span></p></td><td class="c13"><p class="c4"><span>10</span></p></td><td class="c13"><p class="c4"><span>9</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer 10</span><span class="c3">&times;</span><span>H(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>ddH2O(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>7</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span class="c7">Xba</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.s3eqhw38nr24"></a><span>9.16</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.8um0wy4nrt8u"></a><span>Digest 6 and leub</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span>6</span></p></td><td class="c9"><p class="c4"><span>Leu</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c9"><p class="c4"><span>15</span></p></td><td class="c9"><p class="c4"><span>9</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer 10</span><span class="c3">&times;</span><span>M(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>7</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>enzymew</span></p></td><td class="c9"><p class="c4"><span class="c7">Spe</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l) </span><span class="c3">1</span></p></td><td class="c9"><p class="c4"><span class="c7">Xba</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l) 1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l) 1</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.5jlkua87etap"></a><span>9.18</span></h1><h3 class="c4"><a name="h.wll2rnwql4h5"></a><span>Ligate Leu and 6 using 3A</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>Leu</span></p></td><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>buffer</span></p></td><td class="c1"><p class="c4"><span>T4</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>10</span><span class="c3">&micro;</span><span>l</span></p></td><td class="c1"><p class="c4"><span class="c3">2&micro;</span><span>l</span></p></td><td class="c1"><p class="c4"><span class="c3">6&micro;</span><span>l</span></p></td><td class="c1"><p class="c4"><span class="c3">2&micro;</span><span>l</span></p></td><td class="c1"><p class="c4"><span class="c3">1&micro;</span><span>l</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.8d09k2ryw264"></a><span>Gel picture&#65306;</span></h3><p class="c4"><img height="273" src="images/image03.jpg" width="562"></p><h3 class="c4"><a name="h.pzff1yr1mq6n"></a><span>Digest 6</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c4"><span>number</span></p></td><td class="c1"><p class="c4"><span>DNA(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c1"><p class="c4"><span>Buffer 10</span><span class="c3">&times;</span><span>&nbsp;(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c1"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c1"><p class="c4"><span>ddH2O(</span><span class="c3">&micro;</span><span>l)</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>9</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>8</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.rjhja6hena4r"></a><span>9.20</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.doclcz4jf63n"></a><span>Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c38"><p class="c4"><span>number</span></p></td><td class="c50"><p class="c4"><span class="c27 c3">n</span><span class="c27 c7">g/</span><span class="c27 c3">&micro;</span><span class="c27 c7">l</span></p></td><td class="c50"><p class="c4"><span class="c7">A260</span><span class="c3">&#8725;</span><span class="c7">A280</span></p></td></tr><tr class="c12"><td class="c38"><p class="c4"><span>6</span></p></td><td class="c50"><p class="c4"><span>179.5</span></p></td><td class="c50"><p class="c4"><span>1.851</span></p></td></tr><tr class="c12"><td class="c38"><p class="c4"><span>pSB1C3-1</span></p></td><td class="c50"><p class="c4"><span>6.3</span></p></td><td class="c50"><p class="c2"><span></span></p></td></tr><tr class="c12"><td class="c38"><p class="c4"><span>pSB1C3-2</span></p></td><td class="c50"><p class="c4"><span>8.6</span></p></td><td class="c50"><p class="c4"><span>3.510</span></p></td></tr><tr class="c12"><td class="c38"><p class="c4"><span>pSB1C3-3</span></p></td><td class="c50"><p class="c4"><span>5.1</span></p></td><td class="c50"><p class="c2"><span></span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.xbnykbw91qca"></a><span>Digest Leu</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c4"><span>DNA(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c1"><p class="c4"><span>Buffer 10</span><span class="c3">&times;</span><span>M(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c1"><p class="c19 c4"><span>ddH2O(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c1"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c1"><p class="c4"><span class="c7">Xba</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>8</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.ebm9jwpwvzye"></a><span>Ligate Leu&amp; 6</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c4"><span>Leu</span></p></td><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>buffer</span></p></td><td class="c1"><p class="c4"><span>T4</span></p></td><td class="c1"><p class="c4"><span>ddH2O</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>7</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.gx5ptw3fqhxn"></a><span>9.21</span></h1><h3 class="c4"><a name="h.t4pz3r81wrf0"></a><span>Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span class="c27 c3">n</span><span class="c27 c7">g/</span><span class="c27 c3">&micro;</span><span class="c27 c7">l</span></p></td><td class="c9"><p class="c4"><span class="c7">A260</span><span class="c3">&#8725;</span><span class="c7">A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>rai</span><span class="c3">&#8544;</span><span>pro-3</span></p></td><td class="c9"><p class="c4"><span>29.5</span></p></td><td class="c9"><p class="c4"><span>1.666</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>rai</span><span class="c3">&#8544;</span><span>pro-2</span></p></td><td class="c9"><p class="c4"><span>28.5</span></p></td><td class="c9"><p class="c4"><span>1.885</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>rai</span><span class="c3">&#8544;</span><span>pro-1</span></p></td><td class="c9"><p class="c4"><span>23.0</span></p></td><td class="c9"><p class="c4"><span>1.818</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>C0076</span></p></td><td class="c9"><p class="c4"><span>161.0</span></p></td><td class="c9"><p class="c4"><span>1.793</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>E0020e0fp-1</span></p></td><td class="c9"><p class="c4"><span>28.5</span></p></td><td class="c9"><p class="c4"><span>1.676</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>E0020e0fp-2</span></p></td><td class="c9"><p class="c4"><span>19.5</span></p></td><td class="c9"><p class="c4"><span>2.191</span></p></td></tr></tbody></table><h3 class="c29 c4"><a name="h.ahkncaylcg39"></a></h3><h3 class="c4"><a name="h.wwp8s7phrpou"></a><span>Digestion 5</span><span class="c3">&micro;</span><span>l</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c4"><span>number</span></p></td><td class="c5"><p class="c4"><span>rai</span><span class="c3">&#8544;</span><span>pro-1</span></p></td><td class="c13"><p class="c4"><span>rai</span><span class="c3">&#8544;</span><span>pro-2</span></p></td><td class="c13"><p class="c4"><span>rai</span><span class="c3">&#8544;</span><span>pro-3</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DNA(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>4</span></p></td><td class="c13"><p class="c4"><span>4</span></p></td><td class="c13"><p class="c4"><span>4</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer 10</span><span class="c3">&times;</span><span>H (</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>0.5</span></p></td><td class="c13"><p class="c4"><span>0.5</span></p></td><td class="c13"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>EcoRI(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>0.25</span></p></td><td class="c13"><p class="c4"><span>0.25</span></p></td><td class="c13"><p class="c4"><span>0.25</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>PstI(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>0.25</span></p></td><td class="c13"><p class="c4"><span>0.25</span></p></td><td class="c13"><p class="c4"><span>0.25</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.5joce4249z7i"></a><span>20</span><span class="c3">&micro;</span><span>l</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>number</span></p></td><td class="c0"><p class="c4"><span>DNA(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c0"><p class="c4"><span>Buffer 10</span><span class="c3">&times;</span><span>M(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c0"><p class="c4 c19"><span>ddH2O(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c6"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c6"><p class="c4"><span class="c7">Xba</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>C0076</span></p></td><td class="c0"><p class="c4"><span>6.5</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c0"><p class="c4"><span>9.5</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.c6a30hxeop1y"></a><span>50</span><span class="c3">&micro;</span><span>l</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>number</span></p></td><td class="c0"><p class="c4"><span>DNA(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c0"><p class="c4"><span>Buffer 10</span><span class="c3">&times;</span><span>M(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c0"><p class="c19 c4"><span>ddH2O(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c6"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c6"><p class="c4"><span class="c7">Xba</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>E0020</span></p></td><td class="c0"><p class="c4"><span>24</span></p></td><td class="c0"><p class="c4"><span>5</span></p></td><td class="c0"><p class="c4"><span>18</span></p></td><td class="c6"><p class="c4"><span>1.5</span></p></td><td class="c6"><p class="c4"><span>1.5</span></p></td></tr></tbody></table><p class="c2"><span></span></p><p class="c2"><span></span></p><p class="c2"><span></span></p><h1 class="c4"><a name="h.dx3lcv3d11b7"></a><span>9.23</span></h1><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c17"><p class="c4"><span>devive</span></p></td><td class="c23"><p class="c4 c22"><span>&#19968;</span></p></td><td class="c14"><p><span></span></p></td><td class="c43"><p class="c4 c22"><span>&#20108;</span></p></td><td class="c17"><p><span></span></p></td><td class="c17"><p class="c4 c22"><span>&#19977;</span></p></td><td class="c23"><p><span></span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>number</span></p></td><td class="c23"><p class="c4"><span>5-4(AC)</span></p></td><td class="c14"><p class="c4"><span>Leu-6(A)</span></p></td><td class="c43"><p class="c4"><span>18-3-11(AK)</span></p></td><td class="c17"><p class="c4"><span>Leu-6(A)</span></p></td><td class="c17"><p class="c4"><span>Leu-6(A)</span></p></td><td class="c23"><p class="c4"><span>13(A)</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>DNA(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c23"><p class="c4"><span>16</span></p></td><td class="c14"><p class="c4"><span>13.5</span></p></td><td class="c43"><p class="c4"><span>6.5</span></p></td><td class="c17"><p class="c4"><span>13..5</span></p></td><td class="c17"><p class="c4"><span>13.5</span></p></td><td class="c23"><p class="c4"><span>12</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>Buffer (</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c23"><p class="c4"><span>2</span></p></td><td class="c14"><p class="c4"><span>2</span></p></td><td class="c43"><p class="c4"><span>2</span></p></td><td class="c17"><p class="c4"><span>2</span></p></td><td class="c17"><p class="c4"><span>2</span></p></td><td class="c23"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>ddH2O(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c23"><p class="c4"><span>0</span></p></td><td class="c14"><p class="c4"><span>2.5</span></p></td><td class="c43"><p class="c4"><span>9.5</span></p></td><td class="c17"><p class="c4"><span>2.5</span></p></td><td class="c17"><p class="c4"><span>2.5</span></p></td><td class="c23"><p class="c4"><span>4</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>enzyme</span></p></td><td class="c23"><p class="c4"><span>EcoRI1</span></p></td><td class="c14"><p class="c4"><span class="c7">Xba</span><span class="c3">&#8544;1</span></p></td><td class="c43"><p class="c4"><span>EcoRI 1</span></p></td><td class="c17"><p class="c4"><span class="c7">Xba</span><span class="c3">&#8544;1</span></p></td><td class="c17"><p class="c4"><span>EcoRI 1</span></p></td><td class="c23"><p class="c4"><span class="c7">Xba</span><span class="c3">&#8544;1</span></p></td></tr><tr class="c12"><td class="c17"><p class="c2"><span class="c3"></span></p></td><td class="c23"><p class="c4"><span class="c7">Spe</span><span class="c3">&#8544;1</span></p></td><td class="c14"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;1</span></p></td><td class="c43"><p class="c4"><span class="c7">Spe</span><span class="c3">&#8544;1</span></p></td><td class="c17"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;1</span></p></td><td class="c17"><p class="c4"><span class="c7">Spe</span><span class="c3">&#8544;1</span></p></td><td class="c23"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;1</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.z9hgdmqritzr"></a><span>Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><a href="#" name="id.6d6a1369bdb4"></a><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span class="c27 c3">n</span><span class="c27 c7">g/</span><span class="c27 c3">&micro;</span><span class="c27 c7">l</span></p></td><td class="c9"><p class="c4"><span class="c7">A260</span><span class="c3">&#8725;</span><span class="c7">A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>C0076</span></p></td><td class="c9"><p class="c4"><span>21</span></p></td><td class="c9"><p class="c4"><span>2.154</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Clue-6</span></p></td><td class="c9"><p class="c4"><span>72.5</span></p></td><td class="c9"><p class="c4"><span>1.933</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Ce0020</span></p></td><td class="c9"><p class="c4"><span>22</span></p></td><td class="c9"><p class="c4"><span>2.064</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>6</span></p></td><td class="c9"><p class="c4"><span>82</span></p></td><td class="c9"><p class="c4"><span>2.130</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>13</span></p></td><td class="c9"><p class="c4"><span>82</span></p></td><td class="c9"><p class="c4"><span>1.864</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>45</span></p></td><td class="c9"><p class="c4"><span>1.738</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.30jo6d81y85k"></a><span>9.24</span></h1><h3 class="c4"><a name="h.loc88q13v29m"></a><span>First ligation</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c4 c22"><span>number</span></p></td><td class="c10"><p class="c4 c22"><span>1</span></p></td><td class="c10"><p><span></span></p></td><td class="c10"><p class="c4 c22"><span>2</span></p></td><td class="c16"><p><span></span></p></td><td class="c10"><p class="c4 c22"><span>3</span></p></td><td class="c16"><p><span></span></p></td></tr><tr class="c12"><td class="c5"><p class="c4 c22"><span>part</span></p></td><td class="c10"><p class="c4"><span>18-3-11 </span></p></td><td class="c10"><p class="c4"><span>2.5</span></p></td><td class="c10"><p class="c4"><span>13</span></p></td><td class="c16"><p class="c4"><span>2</span></p></td><td class="c10"><p class="c4"><span>C0076</span></p></td><td class="c16"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c10"><p class="c4"><span>Leu-6</span></p></td><td class="c10"><p class="c4"><span>2</span></p></td><td class="c10"><p class="c4"><span>Leu-6</span></p></td><td class="c16"><p class="c4"><span>2</span></p></td><td class="c10"><p class="c4"><span>6</span></p></td><td class="c16"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4 c22"><span>cariier</span></p></td><td class="c10"><p class="c4 c22"><span>AC 3.5</span></p></td><td class="c10"><p><span></span></p></td><td class="c10"><p class="c4 c22"><span>AC 3.5</span></p></td><td class="c16"><p><span></span></p></td><td class="c10"><p class="c2"><span></span></p></td><td class="c16"><p><span></span></p></td></tr><tr class="c12"><td class="c5"><p class="c4 c22"><span>buffer</span></p></td><td class="c10"><p class="c4 c22"><span>2</span></p></td><td class="c10"><p><span></span></p></td><td class="c10"><p class="c4 c22"><span>2</span></p></td><td class="c16"><p><span></span></p></td><td class="c10"><p class="c4 c22"><span>2</span></p></td><td class="c16"><p><span></span></p></td></tr><tr class="c12"><td class="c5"><p class="c4 c22"><span>T4</span></p></td><td class="c10"><p class="c4 c22"><span>1</span></p></td><td class="c10"><p><span></span></p></td><td class="c10"><p class="c4 c22"><span>1</span></p></td><td class="c16"><p><span></span></p></td><td class="c10"><p class="c4 c22"><span>1</span></p></td><td class="c16"><p><span></span></p></td></tr><tr class="c12"><td class="c5"><p class="c4 c22"><span>ddH2O</span></p></td><td class="c10"><p class="c4 c22"><span>9</span></p></td><td class="c10"><p><span></span></p></td><td class="c10"><p class="c4 c22"><span>9.5</span></p></td><td class="c16"><p><span></span></p></td><td class="c10"><p class="c4 c22"><span>9</span></p></td><td class="c16"><p><span></span></p></td></tr></tbody></table><h1 class="c49 c4"><a name="h.mnap6p5gspdc"></a></h1><h1 class="c4"><a name="h.mnap6p5gspdc"></a><span>9.25</span></h1><h3 class="c4"><a name="h.r2p4yasvsr2t"></a><span>Second ligation</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4 c22"><span>number</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p><span></span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c6"><p><span></span></p></td></tr><tr class="c12"><td class="c9"><p class="c4 c22"><span>part</span></p></td><td class="c0"><p class="c4"><span>E0020</span></p></td><td class="c6"><p class="c4"><span>9</span></p></td><td class="c0"><p class="c4"><span>5-4</span></p></td><td class="c6"><p class="c4"><span>4</span></p></td></tr><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c0"><p class="c4"><span>6</span></p></td><td class="c6"><p class="c4"><span>9</span></p></td><td class="c0"><p class="c4"><span>Leu-6</span></p></td><td class="c6"><p class="c4"><span>2.5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4 c22"><span>cariier</span></p></td><td class="c0"><p class="c2"><span></span></p></td><td class="c6"><p><span></span></p></td><td class="c0"><p class="c4"><span>AK 1.5</span></p></td><td class="c6"><p><span></span></p></td></tr><tr class="c12"><td class="c9"><p class="c4 c22"><span>buffer</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c6"><p><span></span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c6"><p><span></span></p></td></tr><tr class="c12"><td class="c9"><p class="c4 c22"><span>T4</span></p></td><td class="c0"><p class="c4"><span>1.5</span></p></td><td class="c6"><p><span></span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p><span></span></p></td></tr><tr class="c12"><td class="c9"><p class="c4 c22"><span>ddH2O</span></p></td><td class="c0"><p class="c4"><span>5.5</span></p></td><td class="c6"><p><span></span></p></td><td class="c0"><p class="c4"><span>9</span></p></td><td class="c6"><p><span></span></p></td></tr></tbody></table><h3 class="c4"><a name="h.wm6ppyi2kw84"></a><span>Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>number</span></p></td><td class="c0"><p><span></span></p></td><td class="c9"><p class="c4"><span class="c27 c3">n</span><span class="c27 c7">g/</span><span class="c27 c3">&micro;</span><span class="c27 c7">l</span></p></td><td class="c9"><p class="c4"><span class="c7">A260</span><span class="c3">&#8725;</span><span class="c7">A280</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>3</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>29</span></p></td><td class="c9"><p class="c4"><span>2.041</span></p></td></tr><tr class="c12"><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>38</span></p></td><td class="c9"><p class="c4"><span>2.171</span></p></td></tr><tr class="c12"><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>35</span></p></td><td class="c9"><p class="c4"><span>2.265</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>E0020</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>69</span></p></td><td class="c9"><p class="c4"><span>1.816</span></p></td></tr><tr class="c12"><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>31</span></p></td><td class="c9"><p class="c4"><span>1.956</span></p></td></tr><tr class="c12"><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>45</span></p></td><td class="c9"><p class="c4"><span>1.957</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>5-4</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>110.5</span></p></td><td class="c9"><p class="c4"><span>1.939</span></p></td></tr><tr class="c12"><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>152.0</span></p></td><td class="c9"><p class="c4"><span>1.900</span></p></td></tr><tr class="c12"><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>76.5</span></p></td><td class="c9"><p class="c4"><span>1.987</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.dlir95pkge4n"></a><span>9.26</span></h3><h3 class="c4"><a name="h.aift0ymnjhti"></a><span>Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span class="c27 c3">n</span><span class="c27 c7">g/</span><span class="c27 c3">&micro;</span><span class="c27 c7">l</span></p></td><td class="c9"><p class="c4"><span class="c7">A260</span><span class="c3">&#8725;</span><span class="c7">A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>14-19</span></p></td><td class="c9"><p class="c4"><span>112.5</span></p></td><td class="c9"><p class="c4"><span>1.875</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>RFP-1</span></p></td><td class="c9"><p class="c4"><span>145.0</span></p></td><td class="c9"><p class="c4"><span>1.859</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>RFP-2</span></p></td><td class="c9"><p class="c4"><span>138.5</span></p></td><td class="c9"><p class="c4"><span>1.884</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>RFP-3</span></p></td><td class="c9"><p class="c4"><span>162.0</span></p></td><td class="c9"><p class="c4"><span>1.873</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.fe703gdi9fmv"></a><span>9.27</span></h3><h3 class="c4"><a name="h.d0esydvlvvow"></a><span>Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span class="c3 c27">n</span><span class="c27 c7">g/</span><span class="c27 c3">&micro;</span><span class="c27 c7">l</span></p></td><td class="c9"><p class="c4"><span class="c7">A260</span><span class="c3">&#8725;</span><span class="c7">A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>C1</span></p></td><td class="c9"><p class="c4"><span>18.0</span></p></td><td class="c9"><p class="c4"><span>2.022</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>C2</span></p></td><td class="c9"><p class="c4"><span>26.0</span></p></td><td class="c9"><p class="c4"><span>2.097</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>C3</span></p></td><td class="c9"><p class="c4"><span>25.0</span></p></td><td class="c9"><p class="c4"><span>2.041</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>C4</span></p></td><td class="c9"><p class="c4"><span>23.0</span></p></td><td class="c9"><p class="c4"><span>1.862</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>rhiR</span></p></td><td class="c9"><p class="c4"><span>18.5</span></p></td><td class="c9"><p class="c4"><span>1.900</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>leuB</span></p></td><td class="c9"><p class="c4"><span>12.1</span></p></td><td class="c9"><p class="c4"><span>2.000</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>14-119-13</span></p></td><td class="c9"><p class="c4"><span>9.6</span></p></td><td class="c9"><p class="c2"><span></span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>RFP</span></p></td><td class="c9"><p class="c4"><span>12.4</span></p></td><td class="c9"><p class="c4"><span>2.041</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.kd1630mtylul"></a><span>9.30</span></h1><h3 class="c4"><a name="h.31j8dlpozyv4"></a><span>1. measure the density of 3 and digest</span></h3><h3 class="c4"><a name="h.31j8dlpozyv4"></a><span>Density &nbsp;(1) 78 ng/ul &nbsp; (2)62ng/ul</span></h3><h3 class="c4"><a name="h.s92naqwi7onn"></a><span>Digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><h3 class="c4"><a name="h.31j8dlpozyv4"></a><span>DNA</span></h3></td><td class="c5"><p class="c4"><span>Buffer</span></p></td><td class="c13"><p class="c4"><span>XbaI</span></p></td><td class="c13"><p class="c4"><span>PstI</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>16</span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.lfmh1js0pg9v"></a><span>2.measure density of 14&amp;19</span></h3><h3 class="c4"><a name="h.lfmh1js0pg9v"></a><span>(1)112ng/ul &nbsp; (2)84ng/ul</span></h3><h3 class="c4"><a name="h.lfmh1js0pg9v"></a><span>10.1</span></h3><h3 class="c4"><a name="h.lfmh1js0pg9v"></a><span>1.measure density</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c4"><span>3.2</span></p></td><td class="c5"><p class="c4"><span>3.3</span></p></td><td class="c13"><p class="c4"><span>C0076&amp;6</span></p></td><td class="c13"><p class="c4"><span>C0076&amp;6</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>273.5</span></p></td><td class="c5"><p class="c4"><span>275</span></p></td><td class="c13"><p class="c4"><span>164</span></p></td><td class="c13"><p class="c4"><span>169</span></p></td></tr></tbody></table><p class="c2"><span></span></p><p class="c2"><span></span></p>
<div><p class="c2 c22"><span></span></p></div><h1 class="c4"><a name="h.u9me3k1zft2"></a><span>7.21</span></h1><h3 class="c4"><a name="h.5c3btudrepao"></a><span>I &nbsp;Prepare medium</span></h3><h3 class="c4"><a name="h.5c3btudrepao"></a><span>1.soc medium</span></h3><h3 class="c4"><a name="h.5c3btudrepao"></a><span>2.LB medium</span></h3><h3 class="c4"><a name="h.5c3btudrepao"></a><span>3.LBliquid medium</span></h3><h3 class="c4"><a name="h.5c3btudrepao"></a><span>II &nbsp;transform 2011 parts &ndash;OUC Order 1 to 3</span></h3><h3 class="c4"><a name="h.5c3btudrepao"></a><span>III &nbsp;pick up a tested single colony</span></h3><p class="c2"><span></span></p><h1 class="c4"><a name="h.tutvix3ykf10"></a><span>7.22</span></h1><h3 class="c4"><a name="h.g2bioc2tx8l7"></a><span>1&#65294;Extract plasmid of the practicing part</span></h3><h3 class="c4"><a name="h.ufnfesh49wep"></a><span>2. Measure density using distilled water as comparison</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c40"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>A260/A280</span></p></td><td class="c13"><p class="c4"><span>A260/A230</span></p></td><td class="c13"><p class="c4"><span>density&#65288;ng/ul&#65289;</span></p></td></tr><tr class="c40"><td class="c5"><p class="c4"><span>1</span></p></td><td class="c5"><p class="c4"><span>1.892</span></p></td><td class="c13"><p class="c4"><span>2.115</span></p></td><td class="c13"><p class="c4"><span>0.035</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>2</span></p></td><td class="c5"><p class="c4"><span>1.841</span></p></td><td class="c13"><p class="c4"><span>1.227</span></p></td><td class="c13"><p class="c4"><span>0.0405</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>3</span></p></td><td class="c5"><p class="c4"><span>2.299</span></p></td><td class="c13"><p class="c4"><span>4.054</span></p></td><td class="c13"><p class="c4"><span>0.030</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.9fgyukkhs06"></a><span>3. Digestion for 3 hours</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c42"><p class="c2"><span></span></p><p class="c2"><span></span></p></td><td class="c32"><p class="c4"><span>1</span></p></td><td class="c32"><p class="c4"><span>2</span></p></td><td class="c32"><p class="c4"><span>3</span></p></td><td class="c32"><p class="c4"><span>4(Single enzyme cut)</span></p><p class="c2"><span></span></p></td></tr><tr class="c12"><td class="c42"><p class="c4"><span>DNA(ul)</span></p></td><td class="c32"><p class="c4"><span>15</span></p></td><td class="c32"><p class="c4"><span>15</span></p></td><td class="c32"><p class="c4"><span>15</span></p></td><td class="c32"><p class="c4"><span>15</span></p></td></tr><tr class="c12"><td class="c42"><p class="c4"><span>Buffer2(ul)</span></p></td><td class="c32"><p class="c4"><span>5</span></p></td><td class="c32"><p class="c4"><span>5</span></p></td><td class="c32"><p class="c4"><span>5</span></p></td><td class="c32"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c42"><p class="c4"><span>Bsa(ul)</span></p></td><td class="c32"><p class="c4"><span>0.5</span></p></td><td class="c32"><p class="c4"><span>0.5</span></p></td><td class="c32"><p class="c4"><span>0.5</span></p></td><td class="c32"><p class="c4"><span>-----</span></p></td></tr><tr class="c12"><td class="c42"><p class="c4"><span>Double-distilled water(ul)</span></p></td><td class="c32"><p class="c4"><span>27.5</span></p></td><td class="c32"><p class="c4"><span>27.5</span></p></td><td class="c32"><p class="c4"><span>27.5</span></p></td><td class="c32"><p class="c4"><span>29</span></p></td></tr><tr class="c12"><td class="c42"><p class="c4"><span>EcoR I(ul)</span></p></td><td class="c32"><p class="c4"><span>1</span></p></td><td class="c32"><p class="c4"><span>1</span></p></td><td class="c32"><p class="c4"><span>1</span></p></td><td class="c32"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c42"><p class="c4"><span>Pst I(ul)</span></p></td><td class="c32"><p class="c4"><span>1</span></p></td><td class="c32"><p class="c4"><span>1</span></p></td><td class="c32"><p class="c4"><span>1</span></p></td><td class="c32"><p class="c4"><span>-------</span></p></td></tr><tr class="c12"><td class="c42"><p class="c4"><span>Time(h)</span></p></td><td class="c32"><p class="c4"><span>3</span></p></td><td class="c32"><p class="c4"><span>3</span></p></td><td class="c32"><p class="c4"><span>3</span></p></td><td class="c32"><p class="c4"><span>3</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.1ruf9mihj3z2"></a><span>4.check by Electrophoresis</span></h3><p class="c4"><span class="c27">1&#12289;2&#12289;3&#65306;double enzyme&#65292;4&#12289;5&#12289;6&#65306;Single enzyme &#65292;7&#65306;plasmid</span></p><p class="c4"><span class="c27">5.transform 7new parts(number:4-10)</span></p><p class="c4"><span class="c27">6.culture bacteria on LB medium</span></p><p class="c4"><span class="c27">7.culture 3 new parts in Shaker</span></p><h1 class="c4"><a name="h.90bsowdqfwvf"></a><span>7.24</span></h1><h3 class="c4"><a name="h.fllej6viiwbz"></a><span>1. Presrvation species of parts No. ,4,5,6,7,8,9,10</span></h3><h3 class="c4"><a name="h.fllej6viiwbz"></a><span>2. Extract plasmid of 4,5,6,7,8,9,10</span></h3><h3 class="c4"><a name="h.fllej6viiwbz"></a><span>3.check through double enzyme Digestion</span></h3><h3 class="c4 c29"><a name="h.fllej6viiwbz"></a></h3><h1 class="c4"><a name="h.60w0ipkcfych"></a><span>7.25</span></h1><h3 class="c4"><a name="h.r7rj1zpq27u6"></a><span>1. Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c37"><p class="c4"><span>5</span></p></td><td class="c37"><p class="c4"><span>0.020&micro;g/&micro;l</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>4</span></p></td><td class="c37"><p class="c4"><span>0.012&micro;g/&micro;l</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>6</span></p></td><td class="c37"><p class="c4"><span>0.020&micro;g/&micro;l</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>2</span></p></td><td class="c37"><p class="c4"><span>0.015&micro;g/&micro;l</span></p></td></tr></tbody></table><p class="c2"><span></span></p><p class="c2"><span></span></p><h3 class="c4"><a name="h.kj1r3fgjzq4o"></a><span>2. Digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>DNA</span></p></td><td class="c1"><p class="c4"><span>25</span></p></td><td class="c1"><p class="c4"><span>30</span></p></td><td class="c1"><p class="c4"><span>25</span></p></td><td class="c1"><p class="c4"><span>25</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Buffer2</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>BSA</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>H2O</span></p></td><td class="c1"><p class="c4"><span>17.5</span></p></td><td class="c1"><p class="c4"><span>12.5</span></p></td><td class="c1"><p class="c4"><span>17.5</span></p></td><td class="c1"><p class="c4"><span>17.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>enzyme1</span></p></td><td class="c1"><p class="c4"><span>EcoR&#8544;1&micro;l</span></p></td><td class="c1"><p class="c4"><span>EcoR&#8544;1&micro;l</span></p></td><td class="c1"><p class="c4"><span>Spe&#8544;1&micro;l</span></p></td><td class="c1"><p class="c4"><span>Xba&#8544;1&micro;l</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Enzyme2</span></p></td><td class="c1"><p class="c4"><span>Spe&#8544;1&micro;l</span></p></td><td class="c1"><p class="c4"><span>Xba&#8544;1&micro;l</span></p></td><td class="c1"><p class="c4"><span>Pst&#8544;1&micro;l</span></p></td><td class="c1"><p class="c4"><span>Pst&#8544;1&micro;l</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.z9jf6h5q8571"></a><span>3.check by Electrophoresis</span></h3><h3 class="c4"><a name="h.z9jf6h5q8571"></a><span>4.gel extraction</span></h3><h3 class="c4"><a name="h.3xuy6enwwjqe"></a><span>5.measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c4"><span>OUC-order</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Density &nbsp;ug/&micro;l</span></p></td><td class="c1"><p class="c4"><span>0.016</span></p></td><td class="c1"><p class="c4"><span>0.021</span></p></td><td class="c1"><p class="c4"><span>0.027</span></p></td><td class="c1"><p class="c4"><span>0.028</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.7t6dpq2z3uet"></a><span>6. Ligate overnight</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Parts</span></p></td><td class="c9"><p class="c4"><span>2&micro;l 5+3&micro;l 4</span></p></td><td class="c9"><p class="c4"><span>3&micro;l 2+3&micro;l 6</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer</span></p></td><td class="c9"><p class="c4"><span>2&micro;l</span></p></td><td class="c9"><p class="c4"><span>2&micro;l</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>enzyme</span></p></td><td class="c9"><p class="c4"><span>1&micro;l</span></p></td><td class="c9"><p class="c4"><span>1&micro;l</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DdH2O</span></p></td><td class="c9"><p class="c4"><span>2&micro;l</span></p></td><td class="c9"><p class="c4"><span>1&micro;l</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.v8m6lz1ufk1s"></a><span>7.28</span></h1><h3 class="c4"><a name="h.3yqybicztq70"></a><span>Repeat the ligation expriments of 7.25</span></h3><h1 class="c4"><a name="h.iezpb3pvvzxm"></a><span>7.29</span></h1><h3 class="c4"><a name="h.2tk3jc38wcfz"></a><span>Extract plasmids of parts No. 11,12,13,14</span></h3><h1 class="c4"><a name="h.igfocjjyx4ru"></a><span>7.30</span></h1><h3 class="c4"><a name="h.i6nzwpfyvvjl"></a><span>Medium has antibiotic resistance problem, re-transform 11\12\13\14</span></h3><h1 class="c4"><a name="h.ug8l8jxvj3n1"></a><span>7.31</span></h1><h3 class="c4"><a name="h.2qt7wbgkde7s"></a><span>1.cultrue bacteria of 11,12,13,14 in LB liquid medium</span></h3><h3 class="c4"><a name="h.2qt7wbgkde7s"></a><span>2. Extract plasmid of 15,16,17,18,19,20,21,22</span></h3><h3 class="c4"><a name="h.2qt7wbgkde7s"></a><span>3. Optimization the Steps of Extracting plasmid</span></h3><h3 class="c4"><a name="h.2qt7wbgkde7s"></a><span>4.Chloramphericol preparing</span></h3><h3 class="c4"><a name="h.2qt7wbgkde7s"></a><span>5. NaAC&rsquo;s DNA </span><span><a class="c45" href="http:///t">concentration</a></span></h3><p class="c2"><span><a class="c45" href="http:///t"></a></span></p><h1 class="c4"><a name="h.tfyl7y9pxkmo"></a><span>8.1 </span></h1><h3 class="c4"><a name="h.bx2edw2baey7"></a><span>1. Extract plasmid of 11-14 and Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>ng/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>11</span></p></td><td class="c9"><p class="c4"><span>12.7</span></p></td><td class="c9"><p class="c4"><span>1.917</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>12</span></p></td><td class="c9"><p class="c4"><span>10.3</span></p></td><td class="c9"><p class="c4"><span>1.981</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>13</span></p></td><td class="c9"><p class="c4"><span>35.5</span></p></td><td class="c9"><p class="c4"><span>1.919</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>14</span></p></td><td class="c9"><p class="c4"><span>15.7</span></p></td><td class="c9"><p class="c4"><span>1.975</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.hvilmpnl4guw"></a><span>2. Extract plasmid of No.5,4,6,2&#65292;15-20 parts</span></h3><h3 class="c4"><a name="h.hvilmpnl4guw"></a><span>3.culture the colony of GFP on LB medium</span></h3><h1 class="c4"><a name="h.9dt18ihmbdii"></a><span>8.2</span></h1><h3 class="c4"><a name="h.e15sf2yo8hvd"></a><span>Measure density of 15-22,2,4,5,6</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>Parts</span></p></td><td class="c9"><p class="c4"><span>&micro;g/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>0.038</span></p></td><td class="c9"><p class="c4"><span>1.875</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>4</span></p></td><td class="c9"><p class="c4"><span>0.019</span></p></td><td class="c9"><p class="c4"><span>1.298</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>5</span></p></td><td class="c9"><p class="c4"><span>0.022</span></p></td><td class="c9"><p class="c4"><span>1.538</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>6</span></p></td><td class="c9"><p class="c4"><span>0.020</span></p></td><td class="c9"><p class="c4"><span>1.702</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>15</span></p></td><td class="c9"><p class="c4"><span>0.012</span></p></td><td class="c9"><p class="c4"><span>1.237</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>16</span></p></td><td class="c9"><p class="c4"><span>0.024</span></p></td><td class="c9"><p class="c4"><span>1.649</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>17</span></p></td><td class="c9"><p class="c4"><span>0.018</span></p></td><td class="c9"><p class="c4"><span>1.731</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18</span></p></td><td class="c9"><p class="c4"><span>0.015</span></p></td><td class="c9"><p class="c4"><span>1.470</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>21</span></p></td><td class="c9"><p class="c4"><span>0.017</span></p></td><td class="c9"><p class="c4"><span>1.538</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.i1axlh3f3s5k"></a><span>8.3</span></h1><h3 class="c4"><a name="h.q8al8jclaxs9"></a><span>1. Concentrate plasmid using alcohol and NaAC</span></h3><h3 class="c4"><a name="h.q8al8jclaxs9"></a><span>2. Measure density after concentrating</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>ng/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>103</span></p></td><td class="c9"><p class="c4"><span>1.807</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>4</span></p></td><td class="c9"><p class="c4"><span>33</span></p></td><td class="c9"><p class="c4"><span>1.811</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>5</span></p></td><td class="c9"><p class="c4"><span>56</span></p></td><td class="c9"><p class="c4"><span>1.836</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>6</span></p></td><td class="c9"><p class="c4"><span>41</span></p></td><td class="c9"><p class="c4"><span>1.745</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.s9gc90q0jnk2"></a><span>8.4</span></h1><h3 class="c4"><a name="h.w2vf5a97h188"></a><span>Digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>Double enzymes</span></p></td><td class="c1"><p><span></span></p></td><td class="c1"><p class="c4"><span>Single enzyme</span></p></td><td class="c1"><p><span></span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Parts</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>6</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>DNA</span></p></td><td class="c1"><p class="c4"><span>25</span></p></td><td class="c1"><p class="c4"><span>13</span></p></td><td class="c1"><p class="c4"><span>27</span></p></td><td class="c1"><p class="c4"><span>25</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Buffer</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>BSA</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>ddH2O</span></p></td><td class="c1"><p class="c4"><span>16.5</span></p></td><td class="c1"><p class="c4"><span>28.5</span></p></td><td class="c1"><p class="c4"><span>16.5</span></p></td><td class="c1"><p class="c4"><span>18.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>enzyme</span></p></td><td class="c1"><p class="c4"><span>EcoR&#8544; 1.5</span></p></td><td class="c1"><p class="c4"><span>Xba&#8544;1.5</span></p></td><td class="c1"><p class="c4"><span>Xba&#8544;1</span></p></td><td class="c1"><p class="c4"><span>Spe&#8544;1</span></p></td></tr><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>Spe&#8544;1.5</span></p></td><td class="c1"><p class="c4"><span>Pst&#8544;1.5</span></p></td><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c2"><span></span></p></td></tr></tbody></table><p class="c2"><span></span></p><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>Parts</span></p></td><td class="c9"><p class="c4"><span>4</span></p></td><td class="c9"><p class="c4"><span>6</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>28.5</span></p></td><td class="c9"><p class="c4"><span>30</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer</span></p></td><td class="c9"><p class="c4"><span>EcoR&#8544;buffer 5</span></p></td><td class="c9"><p class="c4"><span>Buffer3 5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>BSA</span></p></td><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>15.5</span></p></td><td class="c9"><p class="c4"><span>13.5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>enzyme</span></p></td><td class="c9"><p class="c4"><span>EcoR&#8544;1</span></p></td><td class="c9"><p class="c4"><span>Pst&#8544;1</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.hhwrahhkoe43"></a><span>8.5</span></h1><h3 class="c4"><a name="h.t0nrpb7qcvp0"></a><span>1. Begin device 2</span></h3><h3 class="c4"><a name="h.t0nrpb7qcvp0"></a><span>2. Design experimental procedures: </span></h3><h3 class="c4"><a name="h.t0nrpb7qcvp0"></a><span>3. First construction with parts of device 2</span></h3><p class="c2"><span class="c44"></span></p><h1 class="c4"><a name="h.tzzxaggc2un4"></a><span>8.6</span></h1><h3 class="c4"><a name="h.yzze9tnt5vuy"></a><span>2+6 successfully ligate</span></h3><h3 class="c4"><a name="h.8c49qiwztmgq"></a><span>Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>Parts</span></p></td><td class="c9"><p class="c4"><span>&micro;g/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>0.085</span></p></td><td class="c9"><p class="c4"><span>1.910</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>16</span></p></td><td class="c9"><p class="c4"><span>0.119</span></p></td><td class="c9"><p class="c4"><span>1.867</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18</span></p></td><td class="c9"><p class="c4"><span>0.019</span></p></td><td class="c9"><p class="c4"><span>2.033</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.nzti3vckiivz"></a><span>Measure density after concentrating</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>Parts</span></p></td><td class="c9"><p class="c4"><span>&micro;g/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18</span></p></td><td class="c9"><p class="c4"><span>0.0605</span></p></td><td class="c9"><p class="c4"><span>1.921</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>11</span></p></td><td class="c9"><p class="c4"><span>0.0335</span></p></td><td class="c9"><p class="c4"><span>2.107</span></p></td></tr></tbody></table><h3 class="c29 c4"><a name="h.ewnnzrlm06qu"></a></h3><h3 class="c4"><a name="h.pf64q93rtmr3"></a><span>Digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>Double enzymes</span></p></td><td class="c1"><p><span></span></p></td><td class="c1"><p class="c4"><span>Single enzyme</span></p></td><td class="c1"><p><span></span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Parts</span></p></td><td class="c1"><p class="c4"><span>18</span></p></td><td class="c1"><p class="c4"><span>16</span></p></td><td class="c1"><p class="c4"><span>3</span></p></td><td class="c1"><p class="c4"><span>11</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>DNA</span></p></td><td class="c1"><p class="c4"><span>20</span></p></td><td class="c1"><p class="c4"><span>10</span></p></td><td class="c1"><p class="c4"><span>15</span></p></td><td class="c1"><p class="c4"><span>29</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Buffer</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>BSA</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>ddH2O</span></p></td><td class="c1"><p class="c4"><span>21.5</span></p></td><td class="c1"><p class="c4"><span>31.5</span></p></td><td class="c1"><p class="c4"><span>28</span></p></td><td class="c1"><p class="c4"><span>14.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>enzyme</span></p></td><td class="c1"><p class="c4"><span>EcoR&#8544; 1.5</span></p></td><td class="c1"><p class="c4"><span>Xba&#8544;1.5</span></p></td><td class="c1"><p class="c4"><span>Xba&#8544;1</span></p></td><td class="c1"><p class="c4"><span>Spe&#8544;1</span></p></td></tr><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>Spe&#8544;1.5</span></p></td><td class="c1"><p class="c4"><span>Pst&#8544;1.5</span></p></td><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c2"><span></span></p></td></tr></tbody></table><h3 class="c4"><a name="h.id7koc7n89jl"></a><span>After 8hours </span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><h3 class="c4"><a name="h.euom5tw4uac7"></a><span>Parts</span></h3></td><td class="c9"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>11</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>20</span></p></td><td class="c9"><p class="c4"><span>20</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer</span></p></td><td class="c9"><p class="c4"><span>EcoR&#8544;buffer 5</span></p></td><td class="c9"><p class="c4"><span>Buffer3 5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>BSA</span></p></td><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>23.5</span></p></td><td class="c9"><p class="c4"><span>23</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>enzyme</span></p></td><td class="c9"><p class="c4"><span>EcoR&#8544;1</span></p></td><td class="c9"><p class="c4"><span>Pst&#8544;1</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.ekhxnmiid85t"></a><span>8.7</span></h1><h3 class="c4"><a name="h.zb49mmwonyx9"></a><span>1. Checking 4+5, 2+6 &lsquo;s ligation by Gel electrophoresis</span></h3><h3 class="c4"><a name="h.sts8pafwtw56"></a><span>&nbsp;Digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>&mu;L</span></p></td><td class="c9"><p class="c4"><span>2+6</span></p></td><td class="c9"><p class="c4"><span>4+5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>2&#65288;53ng&#65289;</span></p></td><td class="c9"><p class="c4"><span>3(28ng)</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer2</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>BSA</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>6.7</span></p></td><td class="c9"><p class="c4"><span>5.9</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>EcoR&#8544;</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Pst&#8544;</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td></tr></tbody></table><h3 class="c29 c4"><a name="h.7hb0wq9fet7i"></a></h3><h3 class="c4"><a name="h.pxbfcxpcw2h0"></a><span>check by small gel</span></h3><h1 class="c49 c4"><a name="h.1n6cx51ccc1m"></a></h1><h1 class="c4"><a name="h.hzg362nyf3j"></a><span>8.8</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.ms09fpm4vjum"></a><span>1.transform the parts of 3-18,11-16,14-19</span></h3><h3 class="c4"><a name="h.ms09fpm4vjum"></a><span>2. Active bacteria of 4.5.18.11 and culture in LB liquid medium</span></h3><h3 class="c4"><a name="h.ms09fpm4vjum"></a><span>3. Presrvation species parts of 4-5</span></h3><p class="c2"><span></span></p><h1 class="c4"><a name="h.fel77li0jvq3"></a><span>8.9</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.golezolfnu2v"></a><span>1.check 2+6,4+5 by small gel </span></h3><h3 class="c4"><a name="h.golezolfnu2v"></a><span>2. Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>number</span></p></td><td class="c13"><p class="c4"><span>ng/&micro;l</span></p></td><td class="c13"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>2+6</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>58</span></p></td><td class="c13"><p class="c4"><span>1.902</span></p></td></tr><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>78.5</span></p></td><td class="c13"><p class="c4"><span>1.826</span></p></td></tr><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>3</span></p></td><td class="c13"><p class="c4"><span>62.5</span></p></td><td class="c13"><p class="c4"><span>1.953</span></p></td></tr><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>4</span></p></td><td class="c13"><p class="c4"><span>60.5</span></p></td><td class="c13"><p class="c4"><span>2.017</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>4+5</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>31</span></p></td><td class="c13"><p class="c4"><span>2.081</span></p></td></tr><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>33</span></p></td><td class="c13"><p class="c4"><span>2.089</span></p></td></tr><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>3</span></p></td><td class="c13"><p class="c4"><span>20.5</span></p></td><td class="c13"><p class="c4"><span>2.291</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.95qz8jl117p"></a><span>3. Digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c10"><p class="c4"><span>parts</span></p></td><td class="c10"><p class="c4"><span>2+6</span></p></td><td class="c10"><p><span></span></p></td><td class="c10"><p><span></span></p></td><td class="c10"><p><span></span></p></td><td class="c10"><p class="c4"><span>5+4</span></p></td><td class="c16"><p><span></span></p></td><td class="c16"><p><span></span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>number</span></p></td><td class="c10"><p class="c4"><span>1</span></p></td><td class="c10"><p class="c4"><span>2</span></p></td><td class="c10"><p class="c4"><span>3</span></p></td><td class="c10"><p class="c4"><span>4</span></p></td><td class="c10"><p class="c4"><span>1</span></p></td><td class="c16"><p class="c4"><span>2</span></p></td><td class="c16"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>DNA</span></p></td><td class="c10"><p class="c4"><span>2</span></p></td><td class="c10"><p class="c4"><span>2</span></p></td><td class="c10"><p class="c4"><span>2</span></p></td><td class="c10"><p class="c4"><span>2</span></p></td><td class="c10"><p class="c4"><span>3</span></p></td><td class="c16"><p class="c4"><span>3</span></p></td><td class="c16"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>Buffer3</span></p></td><td class="c10"><p class="c4"><span>1</span></p></td><td class="c10"><p class="c4"><span>1</span></p></td><td class="c10"><p class="c4"><span>1</span></p></td><td class="c10"><p class="c4"><span>1</span></p></td><td class="c10"><p class="c4"><span>1</span></p></td><td class="c16"><p class="c4"><span>1</span></p></td><td class="c16"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>BSA</span></p></td><td class="c10"><p class="c4"><span>0.1</span></p></td><td class="c10"><p class="c4"><span>0.1</span></p></td><td class="c10"><p class="c4"><span>0.1</span></p></td><td class="c10"><p class="c4"><span>0.1</span></p></td><td class="c10"><p class="c4"><span>0.1</span></p></td><td class="c16"><p class="c4"><span>0.1</span></p></td><td class="c16"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>ddH2O</span></p></td><td class="c10"><p class="c4"><span>6.5</span></p></td><td class="c10"><p class="c4"><span>6.5</span></p></td><td class="c10"><p class="c4"><span>6.5</span></p></td><td class="c10"><p class="c4"><span>6.5</span></p></td><td class="c10"><p class="c4"><span>5.5</span></p></td><td class="c16"><p class="c4"><span>5.5</span></p></td><td class="c16"><p class="c4"><span>5.5</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>EcoR&#8544;</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c16"><p class="c4"><span>0.2</span></p></td><td class="c16"><p class="c4"><span>0.2</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>Pst&#8544;</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c10"><p class="c4"><span>0.2</span></p></td><td class="c16"><p class="c4"><span>0.2</span></p></td><td class="c16"><p class="c4"><span>0.2</span></p></td></tr></tbody></table><h3 class="c29 c4"><a name="h.ew5rjvlhlbes"></a></h3><h3 class="c4"><a name="h.ew5rjvlhlbes"></a><span>4. Extract plasmid of &nbsp;4,5,18,11</span></h3><h3 class="c4"><a name="h.fbuct55jz3p2"></a><span>Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><h3 class="c4"><a name="h.hqsj9u31i72y"></a><span>number</span></h3></td><td class="c9"><p class="c4"><span>ng/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>5</span></p></td><td class="c9"><p class="c4"><span>73</span></p></td><td class="c9"><p class="c4"><span>2.000</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>4</span></p></td><td class="c9"><p class="c4"><span>71.5</span></p></td><td class="c9"><p class="c4"><span>2.072</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18</span></p></td><td class="c9"><p class="c4"><span>32.5</span></p></td><td class="c9"><p class="c4"><span>2.305</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>85</span></p></td><td class="c9"><p class="c4"><span>1.910</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>11</span></p></td><td class="c9"><p class="c4"><span>95.5</span></p></td><td class="c9"><p class="c4"><span>1.950</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>16</span></p></td><td class="c9"><p class="c4"><span>119</span></p></td><td class="c9"><p class="c4"><span>1.867</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.3z29ux1deqzo"></a><span>Digestion 50&micro;l</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c17"><p class="c2"><span></span></p></td><td class="c17"><p class="c4"><span>5</span></p></td><td class="c21"><p class="c4"><span>4</span></p></td><td class="c17"><p class="c4"><span>18</span></p></td><td class="c21"><p class="c4"><span>3</span></p></td><td class="c21"><p class="c4"><span>11</span></p></td><td class="c21"><p class="c4"><span>16</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>DNA</span></p></td><td class="c17"><p class="c4"><span>13</span></p></td><td class="c21"><p class="c4"><span>13</span></p></td><td class="c17"><p class="c4"><span>30</span></p></td><td class="c21"><p class="c4"><span>11</span></p></td><td class="c21"><p class="c4"><span>10</span></p></td><td class="c21"><p class="c4"><span>8</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>Buffer3</span></p></td><td class="c17"><p class="c4"><span>5</span></p></td><td class="c21"><p class="c4"><span>5</span></p></td><td class="c17"><p class="c4"><span>5</span></p></td><td class="c21"><p class="c4"><span>5</span></p></td><td class="c21"><p class="c4"><span>5</span></p></td><td class="c21"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>BSA</span></p></td><td class="c17"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td><td class="c17"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>ddH2O</span></p></td><td class="c17"><p class="c4"><span>29.5</span></p></td><td class="c21"><p class="c4"><span>29.5</span></p></td><td class="c17"><p class="c4"><span>19.5</span></p></td><td class="c21"><p class="c4"><span>31.5</span></p></td><td class="c21"><p class="c4"><span>30.5</span></p></td><td class="c21"><p class="c4"><span>34.5</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>enzyme</span></p></td><td class="c17"><p class="c4"><span>EcoR&#8544; 1</span></p></td><td class="c21"><p class="c4"><span>Xba&#8544;1</span></p></td><td class="c17"><p class="c4"><span>EcoR&#8544; 1</span></p></td><td class="c21"><p class="c4"><span>Xba&#8544;1</span></p></td><td class="c21"><p class="c4"><span>EcoR&#8544; 1</span></p></td><td class="c21"><p class="c4"><span>Xba&#8544;1</span></p></td></tr><tr class="c12"><td class="c17"><p class="c2"><span></span></p></td><td class="c17"><p class="c4"><span>Spe&#8544;1</span></p></td><td class="c21"><p class="c4"><span>Pst&#8544;1</span></p></td><td class="c17"><p class="c4"><span>Spe&#8544;1</span></p></td><td class="c21"><p class="c4"><span>Pst&#8544;1</span></p></td><td class="c21"><p class="c4"><span>Spe&#8544;1</span></p></td><td class="c21"><p class="c4"><span>Pst&#8544;1</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.jmqfpq9xkm9z"></a><span>8.11</span></h1><h4 class="c4"><a name="h.m6rgdwzdsd3p"></a><span>1. Checking ligation correctness of 14&amp;19 using small gel----fails</span></h4><h4 class="c4"><a name="h.m6rgdwzdsd3p"></a><span>2.try to ligate using 3A</span></h4><h1 class="c4"><a name="h.z0x6hx6e7ltc"></a><span>8.12</span></h1><h3 class="c4"><a name="h.dm8f9683o5dr"></a><span>1. Extract plasmid of &nbsp;5+4 and Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span>ng/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>55</span></p></td><td class="c9"><p class="c4"><span>1.964</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>39</span></p></td><td class="c9"><p class="c4"><span>2.324</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>41</span></p></td><td class="c9"><p class="c4"><span>2.412</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>4</span></p></td><td class="c9"><p class="c4"><span>81</span></p></td><td class="c9"><p class="c4"><span>2.051</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>5</span></p></td><td class="c9"><p class="c4"><span>42</span></p></td><td class="c9"><p class="c4"><span>2.154</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>6</span></p></td><td class="c9"><p class="c4"><span>58</span></p></td><td class="c9"><p class="c4"><span>1.966</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.77mylrq8vnt2"></a><span>2.check by single enzyme digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c17"><p class="c4"><span>number</span></p></td><td class="c17"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>2</span></p></td><td class="c17"><p class="c4"><span>3</span></p></td><td class="c21"><p class="c4"><span>4</span></p></td><td class="c21"><p class="c4"><span>5</span></p></td><td class="c21"><p class="c4"><span>6</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>DNA</span></p></td><td class="c17"><p class="c4"><span>3</span></p></td><td class="c21"><p class="c4"><span>4</span></p></td><td class="c17"><p class="c4"><span>4</span></p></td><td class="c21"><p class="c4"><span>2</span></p></td><td class="c21"><p class="c4"><span>4</span></p></td><td class="c21"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>Buffer EcoR I</span></p></td><td class="c17"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c17"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>ddH2O</span></p></td><td class="c17"><p class="c4"><span>5.5</span></p></td><td class="c21"><p class="c4"><span>4.5</span></p></td><td class="c17"><p class="c4"><span>4.5</span></p></td><td class="c21"><p class="c4"><span>6.5</span></p></td><td class="c21"><p class="c4"><span>4.5</span></p></td><td class="c21"><p class="c4"><span>5.5</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>EcoR&#8544;</span></p></td><td class="c17"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td><td class="c17"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td></tr></tbody></table><h3 class="c29 c4"><a name="h.sp1o9aob8jry"></a></h3><h3 class="c4"><a name="h.f568ba30zhy7"></a><span>Check using small gel</span></h3><h3 class="c4"><a name="h.f568ba30zhy7"></a><span>3.cultrue bacteria of part 13</span></h3><h3 class="c4"><a name="h.f568ba30zhy7"></a><span>4. ligate &nbsp;2+6+3,11+16,18+13</span></h3><p class="c2"><span></span></p><h1 class="c4"><a name="h.alf83syip56z"></a><span>8.13</span></h1><h3 class="c4"><a name="h.ke3ebgbum5n"></a><span>1.Extract plasmid </span></h3><h3 class="c4"><a name="h.ke3ebgbum5n"></a><span>2.pick colony of 2+6+3,18+3,16+3 and culture in shaker</span></h3><p class="c2"><span></span></p><h1 class="c4"><a name="h.e0ipvrwodbd7"></a><span>8.14 </span></h1><h4 class="c4"><a name="h.qq0p6vx7yo2i"></a><span>Checking whether colony of different size contain different plasmids, or whether could be used to pick colony raising transformation possibility. But it made no difference</span></h4><h1 class="c4"><a name="h.5k95r8qmtjok"></a><span>8.15</span></h1><h3 class="c4"><a name="h.wy8969l7pop"></a><span>1.measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span>&micro;g/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>2+6+3-1</span></p></td><td class="c9"><p class="c4"><span>0.032</span></p></td><td class="c9"><p class="c4"><span>3.439</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>2+6+3-2</span></p></td><td class="c9"><p class="c4"><span>0.064</span></p></td><td class="c9"><p class="c4"><span>2.370</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>11+16</span></p></td><td class="c9"><p class="c4"><span>0.056</span></p></td><td class="c9"><p class="c4"><span>2.306</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>11+16</span></p></td><td class="c9"><p class="c4"><span>0.102</span></p></td><td class="c9"><p class="c4"><span>2.010</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18+3</span></p></td><td class="c9"><p class="c4"><span>0.037</span></p></td><td class="c9"><p class="c4"><span>2.434</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18+3</span></p></td><td class="c9"><p class="c4"><span>0.033</span></p></td><td class="c9"><p class="c4"><span>2.332</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.3m4xfb4u4qv8"></a><span>2. Digestion</span></h3><p class="c2"><span></span></p><h3 class="c4"><a name="h.c1ekew4tc9t5"></a><span>Single enzyme 10&micro;l</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c17"><p class="c4"><span>number</span></p></td><td class="c17"><p class="c4"><span>2+6+3-1</span></p></td><td class="c21"><p class="c4"><span>2+6+3-2</span></p></td><td class="c17"><p class="c4"><span>3+18-1</span></p></td><td class="c21"><p class="c4"><span>3+18-2</span></p></td><td class="c21"><p class="c4"><span>11+16</span></p></td><td class="c21"><p class="c4"><span>11+16</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>DNA</span></p></td><td class="c17"><p class="c4"><span>4</span></p></td><td class="c21"><p class="c4"><span>2</span></p></td><td class="c17"><p class="c4"><span>4</span></p></td><td class="c21"><p class="c4"><span>6</span></p></td><td class="c21"><p class="c4"><span>2.5</span></p></td><td class="c21"><p class="c4"><span>1.5</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>Buffer EcoR I</span></p></td><td class="c17"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c17"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>ddH2O</span></p></td><td class="c17"><p class="c4"><span>4.7</span></p></td><td class="c21"><p class="c4"><span>6.7</span></p></td><td class="c17"><p class="c4"><span>4.7</span></p></td><td class="c21"><p class="c4"><span>2.7</span></p></td><td class="c21"><p class="c4"><span>6.2</span></p></td><td class="c21"><p class="c4"><span>7.2</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>EcoR&#8544;</span></p></td><td class="c17"><p class="c4"><span>0.3</span></p></td><td class="c21"><p class="c4"><span>0.3</span></p></td><td class="c17"><p class="c4"><span>0.3</span></p></td><td class="c21"><p class="c4"><span>0.3</span></p></td><td class="c21"><p class="c4"><span>0.3</span></p></td><td class="c21"><p class="c4"><span>0.3</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.apo7y8k82owr"></a><span>Double enzyme 10&micro;l</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c11"><p class="c4"><span>number</span></p></td><td class="c20"><p class="c4"><span>18+3-1</span></p></td><td class="c11"><p class="c4"><span>18+3-2</span></p></td><td class="c11"><p class="c4"><span>18</span></p></td><td class="c11"><p class="c4"><span>2+6+3-1</span></p></td><td class="c20"><p class="c4"><span>2+6+3-2</span></p></td><td class="c20"><p class="c4"><span>2+6</span></p></td><td class="c20"><p class="c4"><span>11+16</span></p></td><td class="c20"><p class="c4"><span>11+16</span></p></td></tr><tr class="c12"><td class="c11"><p class="c4"><span>DNA</span></p></td><td class="c20"><p class="c4"><span>4</span></p></td><td class="c11"><p class="c4"><span>6</span></p></td><td class="c11"><p class="c4"><span>6</span></p></td><td class="c11"><p class="c4"><span>4</span></p></td><td class="c20"><p class="c4"><span>2</span></p></td><td class="c20"><p class="c4"><span>3</span></p></td><td class="c20"><p class="c4"><span>2.5</span></p></td><td class="c20"><p class="c4"><span>1.5</span></p></td></tr><tr class="c12"><td class="c11"><p class="c4"><span>Buffer3</span></p></td><td class="c20"><p class="c4"><span>1</span></p></td><td class="c11"><p class="c4"><span>1</span></p></td><td class="c11"><p class="c4"><span>1</span></p></td><td class="c11"><p class="c4"><span>1</span></p></td><td class="c20"><p class="c4"><span>1</span></p></td><td class="c20"><p class="c4"><span>1</span></p></td><td class="c20"><p class="c4"><span>1</span></p></td><td class="c20"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c11"><p class="c4"><span>BSA</span></p></td><td class="c20"><p class="c4"><span>0.1</span></p></td><td class="c11"><p class="c4"><span>0.1</span></p></td><td class="c11"><p class="c4"><span>0.1</span></p></td><td class="c11"><p class="c4"><span>0.1</span></p></td><td class="c20"><p class="c4"><span>0.1</span></p></td><td class="c20"><p class="c4"><span>0.1</span></p></td><td class="c20"><p class="c4"><span>0.1</span></p></td><td class="c20"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c11"><p class="c4"><span>ddH2O</span></p></td><td class="c20"><p class="c4"><span>4.3</span></p></td><td class="c11"><p class="c4"><span>2.3</span></p></td><td class="c11"><p class="c4"><span>2.4</span></p></td><td class="c11"><p class="c4"><span>4.3</span></p></td><td class="c20"><p class="c4"><span>6.3</span></p></td><td class="c20"><p class="c4"><span>5.3</span></p></td><td class="c20"><p class="c4"><span>5.8</span></p></td><td class="c20"><p class="c4"><span>6.8</span></p></td></tr><tr class="c12"><td class="c11"><p class="c4"><span>EcoR&#8544;</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c11"><p class="c4"><span>0.3</span></p></td><td class="c11"><p class="c4"><span>0.3</span></p></td><td class="c11"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td></tr><tr class="c12"><td class="c11"><p class="c4"><span>Pst&#8544;</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c11"><p class="c4"><span>0.3</span></p></td><td class="c11"><p class="c4"><span>0.3</span></p></td><td class="c11"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td><td class="c20"><p class="c4"><span>0.3</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.l40msbsdoxxo"></a><span>8.16</span></h1><h3 class="c4"><a name="h.9a0lm2k3wig9"></a><span>Check yesterdays&rsquo; digestion by gel electrophoresis</span></h3><p class="c4"><img height="573" src="images/image00.jpg" width="573"></p><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c47"><p class="c4 c22"><span>Ladder</span></p></td><td class="c48"><p class="c4 c22"><span>18-3</span></p></td><td class="c47"><p><span></span></p></td><td class="c47"><p class="c4 c22"><span>18-3</span></p></td><td class="c47"><p><span></span></p></td><td class="c48"><p class="c4 c22"><span>18</span></p></td><td class="c48"><p class="c4 c22"><span>6-2-3</span></p></td><td class="c48"><p class="c4 c22"><span>6-2-3</span></p></td><td class="c48"><p class="c4 c22"><span>6-2</span></p></td></tr><tr class="c12"><td class="c47"><p class="c4 c22"><span>100bp plus</span></p></td><td class="c48"><p class="c4 c22"><span>single</span></p></td><td class="c47"><p class="c4 c22"><span>double</span></p></td><td class="c47"><p class="c4 c22"><span>single</span></p></td><td class="c47"><p class="c4 c22"><span>double</span></p></td><td class="c48"><p class="c4 c22"><span>double</span></p></td><td class="c48"><p class="c4 c22"><span>double</span></p></td><td class="c48"><p class="c4 c22"><span>double</span></p></td><td class="c48"><p class="c4 c22"><span>double</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.6vm4ek23k6ec"></a><span>8.17</span></h1><h3 class="c4"><a name="h.t0sbfske940j"></a><span>1. Presrvation species of pSB1AC3_BBa13017</span></h3><h3 class="c4"><a name="h.n83ej944zlf8"></a><span>2. Extract plasmid of 18&#65292;pSB1AC3_BBa13017&#65292;measure density </span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span>&micro;g/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-1</span></p></td><td class="c9"><p class="c4"><span>0.199</span></p></td><td class="c9"><p class="c4"><span>2.036</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-2</span></p></td><td class="c9"><p class="c4"><span>0.168</span></p></td><td class="c9"><p class="c4"><span>2.036</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-3</span></p></td><td class="c9"><p class="c4"><span>0.113</span></p></td><td class="c9"><p class="c4"><span>2.083</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-4</span></p></td><td class="c9"><p class="c4"><span>0.103</span></p></td><td class="c9"><p class="c4"><span>2.102</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-5</span></p></td><td class="c9"><p class="c4"><span>0.174</span></p></td><td class="c9"><p class="c4"><span>2.129</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-6</span></p></td><td class="c9"><p class="c4"><span>0.147</span></p></td><td class="c9"><p class="c4"><span>2.115</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-7</span></p></td><td class="c9"><p class="c4"><span>0.064</span></p></td><td class="c9"><p class="c4"><span>2.345</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-8</span></p></td><td class="c9"><p class="c4"><span>0.106</span></p></td><td class="c9"><p class="c4"><span>2.186</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-9</span></p></td><td class="c9"><p class="c4"><span>0.163</span></p></td><td class="c9"><p class="c4"><span>2.138</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1AC3_BBa13017-10</span></p></td><td class="c9"><p class="c4"><span>0.170</span></p></td><td class="c9"><p class="c4"><span>2.092</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18-1</span></p></td><td class="c9"><p class="c4"><span>0.110</span></p></td><td class="c9"><p class="c4"><span>2.095</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18-2</span></p></td><td class="c9"><p class="c4"><span>0.124</span></p></td><td class="c9"><p class="c4"><span>2.102</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>18-3</span></p></td><td class="c9"><p class="c4"><span>0.140</span></p></td><td class="c9"><p class="c4"><span>2.121</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.h74qm3ioftn6"></a><span>3. Prepare medium &nbsp;of &nbsp;TY&#65288;culturing rhizobium&#65289;,CM&#65288;supplemented medium&#65289;</span></h3><h3 class="c4"><a name="h.h74qm3ioftn6"></a><span>4. Digest pSB1AC3_BBa13017 as Carrier</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c37"><p class="c4"><span>DNA</span></p></td><td class="c37"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>Buffer2</span></p></td><td class="c37"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>BSA</span></p></td><td class="c37"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>ddH2O</span></p></td><td class="c37"><p class="c4"><span>37.5</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>EcoR&#8544;</span></p></td><td class="c37"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>Pst&#8544;</span></p></td><td class="c37"><p class="c4"><span>1</span></p></td></tr></tbody></table><p class="c4"><img height="610" src="images/image01.jpg" width="610"></p><h6 class="c4"><a name="h.jnl1gq2qm9xp"></a><span>Extract 3055bp strip</span></h6><h1 class="c4"><a name="h.bgliaogofoy0"></a><span>8.18</span></h1><h3 class="c4"><a name="h.8u5x2xe0ixdw"></a><span>1.Ligate 5,4</span></h3><h3 class="c4"><a name="h.8u5x2xe0ixdw"></a><span>2.transform &nbsp;pSB1C3</span></h3><h3 class="c4"><a name="h.8u5x2xe0ixdw"></a><span>8.19</span></h3><h3 class="c4"><a name="h.8u5x2xe0ixdw"></a><span>1.culture rhizobium on TY medium</span></h3><h3 class="c4"><a name="h.8u5x2xe0ixdw"></a><span>2.prepare CM,M9 medium</span></h3><h3 class="c4"><a name="h.8u5x2xe0ixdw"></a><span>3.LeuB PCR</span></h3><h3 class="c4"><a name="h.8u5x2xe0ixdw"></a><span>4. Presrvation species of Bean strain</span></h3><h3 class="c4"><a name="h.wuw8mhckhe1z"></a><span>5. Extract plasmid and Digest</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c10"><p class="c4"><span>number</span></p></td><td class="c10"><p class="c4"><span>18</span></p></td><td class="c10"><p class="c4"><span>3</span></p></td><td class="c10"><p class="c4"><span>2+6</span></p></td><td class="c10"><p class="c4"><span>16</span></p></td><td class="c10"><p class="c4"><span>11</span></p></td><td class="c16"><p class="c4"><span>14+19</span></p></td><td class="c16"><p class="c4"><span>13</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>DNA</span></p></td><td class="c10"><p class="c4"><span>12</span></p></td><td class="c10"><p class="c4"><span>10</span></p></td><td class="c10"><p class="c4"><span>18</span></p></td><td class="c10"><p class="c4"><span>9</span></p></td><td class="c10"><p class="c4"><span>7</span></p></td><td class="c16"><p class="c4"><span>7</span></p></td><td class="c16"><p class="c4"><span>6</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>Buffer2</span></p></td><td class="c10"><p class="c4"><span>5</span></p></td><td class="c10"><p class="c4"><span>5</span></p></td><td class="c10"><p class="c4"><span>5</span></p></td><td class="c10"><p class="c4"><span>5</span></p></td><td class="c10"><p class="c4"><span>5</span></p></td><td class="c16"><p class="c4"><span>5</span></p></td><td class="c16"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>BSA</span></p></td><td class="c10"><p class="c4"><span>0.5</span></p></td><td class="c10"><p class="c4"><span>0.5</span></p></td><td class="c10"><p class="c4"><span>0.5</span></p></td><td class="c10"><p class="c4"><span>0.5</span></p></td><td class="c10"><p class="c4"><span>0.5</span></p></td><td class="c16"><p class="c4"><span>0.5</span></p></td><td class="c16"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>ddH2O</span></p></td><td class="c10"><p class="c4"><span>30.1</span></p></td><td class="c10"><p class="c4"><span>31.5</span></p></td><td class="c10"><p class="c4"><span>24.1</span></p></td><td class="c10"><p class="c4"><span>33.1</span></p></td><td class="c10"><p class="c4"><span>35.1</span></p></td><td class="c16"><p class="c4"><span>35.1</span></p></td><td class="c16"><p class="c4"><span>36.1</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>enzyme</span></p></td><td class="c10"><p class="c4"><span>EcoRI1.2</span></p></td><td class="c10"><p class="c4"><span>Xba&#8544;1.2</span></p></td><td class="c10"><p class="c4"><span>EcoRI1.2</span></p></td><td class="c10"><p class="c4"><span>Xba&#8544;1.2</span></p></td><td class="c10"><p class="c4"><span>EcoRI1.2</span></p></td><td class="c16"><p class="c4"><span>EcoRI1.2</span></p></td><td class="c16"><p class="c4"><span>Xba&#8544;1.2</span></p></td></tr><tr class="c12"><td class="c10"><p class="c2"><span></span></p></td><td class="c10"><p class="c4"><span>Spe&#8544;1.2</span></p></td><td class="c10"><p class="c4"><span>Pst&#8544;1.2</span></p></td><td class="c10"><p class="c4"><span>Spe&#8544;1.2</span></p></td><td class="c10"><p class="c4"><span>Pst&#8544;1.2</span></p></td><td class="c10"><p class="c4"><span>Spe&#8544;1.2</span></p></td><td class="c16"><p class="c4"><span>Spe&#8544;1.2</span></p></td><td class="c16"><p class="c4"><span>Pst&#8544;1.2</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.79kmvmm53rae"></a><span>8.20</span></h1><h3 class="c4"><a name="h.rb7ae2jhzsxo"></a><span>1. Presrvation species of 5+4 and extract plasmid,measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span>ng/&micro;l</span></p></td><td class="c9"><p class="c4"><span>A260&#8725;A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>99.5</span></p></td><td class="c9"><p class="c4"><span>2.098</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>140.5</span></p></td><td class="c9"><p class="c4"><span>2.162</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>99</span></p></td><td class="c9"><p class="c4"><span>2.152</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>4</span></p></td><td class="c9"><p class="c4"><span>123.5</span></p></td><td class="c9"><p class="c4"><span>2.093</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>5</span></p></td><td class="c9"><p class="c4"><span>137.5</span></p></td><td class="c9"><p class="c4"><span>2.083</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.cd31d7gjoaj9"></a><span>2. Digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>number</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c6"><p class="c4"><span>4</span></p></td><td class="c6"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>DNA</span></p></td><td class="c0"><p class="c4"><span>1.3</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>1.3</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>Buffer3</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>BSA</span></p></td><td class="c0"><p class="c4"><span>0.1</span></p></td><td class="c0"><p class="c4"><span>0.1</span></p></td><td class="c0"><p class="c4"><span>0.1</span></p></td><td class="c6"><p class="c4"><span>0.1</span></p></td><td class="c6"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>ddH2O</span></p></td><td class="c0"><p class="c4"><span>7.1</span></p></td><td class="c0"><p class="c4"><span>7.4</span></p></td><td class="c0"><p class="c4"><span>7.1</span></p></td><td class="c6"><p class="c4"><span>7.4</span></p></td><td class="c6"><p class="c4"><span>7.4</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>EcoR&#8544;</span></p></td><td class="c0"><p class="c4"><span>0.25</span></p></td><td class="c0"><p class="c4"><span>0.25</span></p></td><td class="c0"><p class="c4"><span>0.25</span></p></td><td class="c6"><p class="c4"><span>0.25</span></p></td><td class="c6"><p class="c4"><span>0.25</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>Pst&#8544;</span></p></td><td class="c0"><p class="c4"><span>0.25</span></p></td><td class="c0"><p class="c4"><span>0.25</span></p></td><td class="c0"><p class="c4"><span>0.25</span></p></td><td class="c6"><p class="c4"><span>0.25</span></p></td><td class="c6"><p class="c4"><span>0.25</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c49 c4"><a name="h.wlf6j8rjal3t"></a></h1><h1 class="c4"><a name="h.urk4as897tek"></a><span>8.22</span></h1><h3 class="c4"><a name="h.dwwnj6olsp9f"></a><span>1&#12289;Presrvation species of 5+4 and extract plasmid,measure density, digest</span></h3><h3 class="c4"><a name="h.4k6v2rq1fzij"></a><span>2&#12289;pick single colonies of 19-14&amp;13&#65307;2-6&amp;3 , 11&amp;16 and culture in shaker</span></h3><h3 class="c4"><a name="h.k3thznec7nhf"></a><span>3&#12289;PCR Leu&#65288;50ul&#65289;&#65292;recover and check</span></h3><p class="c2"><span></span></p><p class="c4"><img height="197" src="images/image04.jpg" width="187"></p><p class="c4"><span>1</span><span class="c3">0&times;</span><span>Taq Buffer(with MgSO4) 5&mu;L</span></p><p class="c4"><span>MgCl2 &nbsp;4&mu;L</span></p><p class="c4"><span>Primer-F 1.5&mu;L</span></p><p class="c4"><span>Primer-R 1.5&mu;L</span></p><p class="c4"><span>H2O 33.5&mu;L</span></p><p class="c4"><span>dNTP 2&mu;L</span></p><p class="c4"><span>DNA 2&mu;L</span></p><p class="c4"><span>Taq 0.25&mu;L</span></p><p class="c4"><span>Pfu 0.25&mu;L &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span></p><p class="c4 c22"><span>Recommended By Advicer Tan</span></p><p class="c4 c22"><span>Result: success</span></p><p class="c2"><span></span></p><p class="c2"><span></span></p><p class="c2"><span></span></p><p class="c2"><span></span></p><h1 class="c49 c4"><a name="h.2yfk6buo0k2e"></a></h1><h1 class="c4"><a name="h.2yfk6buo0k2e"></a><span>8.23</span></h1><h3 class="c4"><a name="h.g493ims3jbp8"></a><span>1&#12289;Presrvation species of 2 6&amp;3&#65292;11&amp;16&#65292;14 19&amp;13 and extract plasmid, measure density&#65292;digest</span></h3><h3 class="c4"><a name="h.tknykw38j7c4"></a><span>density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c15"><p class="c4"><span>number</span></p></td><td class="c30"><p class="c4"><span>2 6&amp;3</span></p></td><td class="c54"><p class="c4"><span>11&amp;16</span></p></td><td class="c51"><p class="c4"><span>1419&amp;13</span><span class="c3">&#61569;</span></p></td><td class="c41"><p class="c4"><span class="c3">&#61570;</span></p></td><td class="c30"><p class="c4"><span class="c3">&#61571;</span></p></td><td class="c39"><p class="c4"><span class="c3">&#61572;</span></p></td><td class="c39"><p class="c4"><span class="c3">&#61573;</span></p></td></tr><tr class="c12"><td class="c15"><p class="c4"><span>density</span></p><p class="c4"><span>&#65288;ng/ul&#65289;</span></p></td><td class="c30"><p class="c4"><span>30</span></p></td><td class="c54"><p class="c4"><span>49</span></p></td><td class="c51"><p class="c4"><span>45</span></p></td><td class="c41"><p class="c4"><span>132</span></p></td><td class="c30"><p class="c4"><span>62</span></p></td><td class="c39"><p class="c4"><span>42</span></p></td><td class="c39"><p class="c4"><span>64</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.r1xl2xgbvvgi"></a><span>Digestion</span></h3><h3 class="c4"><a name="h.qvlzmnr7s30"></a><span>Single enzyme</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c30"><p class="c2"><span></span></p></td><td class="c31"><p class="c4"><span>2 6&amp;3</span></p></td><td class="c30"><p class="c4"><span>11&amp;16</span></p></td><td class="c35"><p class="c4"><span>1419&amp;13</span><span class="c3">&#61569;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61570;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61571;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61572;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61573;</span></p></td></tr><tr class="c12"><td class="c30"><p class="c4"><span>DNA</span></p></td><td class="c31"><p class="c4"><span>3.3</span></p></td><td class="c30"><p class="c4"><span>2</span></p></td><td class="c35"><p class="c4"><span>2.2</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1.5</span></p></td><td class="c8"><p class="c4"><span>2.3</span></p></td><td class="c8"><p class="c4"><span>1.5</span></p></td></tr><tr class="c12"><td class="c30"><p class="c4"><span>Buffer4</span></p></td><td class="c31"><p class="c4"><span>1</span></p></td><td class="c30"><p class="c4"><span>1</span></p></td><td class="c35"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c30"><p class="c4"><span>DdH2O</span></p></td><td class="c31"><p class="c4"><span>5.4</span></p></td><td class="c30"><p class="c4"><span>6.6</span></p></td><td class="c35"><p class="c4"><span>6.4</span></p></td><td class="c8"><p class="c4"><span>7.6</span></p></td><td class="c8"><p class="c4"><span>7.1</span></p></td><td class="c8"><p class="c4"><span>6.3</span></p></td><td class="c8"><p class="c4"><span>7.1</span></p></td></tr><tr class="c12"><td class="c30"><p class="c4"><span>XbaI</span></p></td><td class="c31"><p class="c4"><span>0.3</span></p></td><td class="c30"><p class="c4"><span>0.3</span></p></td><td class="c35"><p class="c4"><span>0.3</span></p></td><td class="c8"><p class="c4"><span>0.3</span></p></td><td class="c8"><p class="c4"><span>0.3</span></p></td><td class="c8"><p class="c4"><span>0.3</span></p></td><td class="c8"><p class="c4"><span>0.3</span></p></td></tr><tr class="c12"><td class="c30"><p class="c4"><span>BSA</span></p></td><td class="c31"><p class="c4"><span>0.1</span></p></td><td class="c30"><p class="c4"><span>0.1</span></p></td><td class="c35"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.78jdujd9b23x"></a><span>Double enzymes</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c33"><p class="c2"><span></span></p></td><td class="c8"><p class="c4"><span>2 6&amp;3</span></p></td><td class="c25"><p class="c4"><span>11&amp;16</span></p></td><td class="c35"><p class="c4"><span>1419&amp;13</span><span class="c3">&#61569;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61570;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61571;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61572;</span></p></td><td class="c8"><p class="c4"><span class="c3">&#61573;</span></p></td></tr><tr class="c12"><td class="c33"><p class="c4"><span>DNA</span></p></td><td class="c8"><p class="c4"><span>3.3</span></p></td><td class="c25"><p class="c4"><span>2</span></p></td><td class="c35"><p class="c4"><span>2.2</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1.5</span></p></td><td class="c8"><p class="c4"><span>2.3</span></p></td><td class="c8"><p class="c4"><span>1.5</span></p></td></tr><tr class="c12"><td class="c33"><p class="c4"><span>Buffer4</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c25"><p class="c4"><span>1</span></p></td><td class="c35"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td><td class="c8"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c33"><p class="c4"><span>BSA</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td><td class="c25"><p class="c4"><span>0.1</span></p></td><td class="c35"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td><td class="c8"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c33"><p class="c4"><span>H2O</span></p></td><td class="c8"><p class="c4"><span>5.1</span></p></td><td class="c25"><p class="c4"><span>6.4</span></p></td><td class="c35"><p class="c4"><span>6.2</span></p></td><td class="c8"><p class="c4"><span>7.4</span></p></td><td class="c8"><p class="c4"><span>6.9</span></p></td><td class="c8"><p class="c4"><span>6.1</span></p></td><td class="c8"><p class="c4"><span>6.9</span></p></td></tr><tr class="c12"><td class="c33"><p class="c4"><span>EcoRI-HF</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c25"><p class="c4"><span>0.25</span></p></td><td class="c35"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td></tr><tr class="c12"><td class="c33"><p class="c4"><span>SpeI</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c25"><p class="c4"><span>0.25</span></p></td><td class="c35"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td><td class="c8"><p class="c4"><span>0.25</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.uosio89ahtai"></a><span>result&#65306;success</span></h3><h3 class="c4"><a name="h.oapjx65rnz4x"></a><span>2&#12289;</span><span class="c36">Preservation</span><span>&nbsp;species of CFN42 WT and BL21</span></h3><h3 class="c4"><a name="h.oapjx65rnz4x"></a><span>3&#12289;check 18&amp;3 be small gel &mdash;&mdash;succeed</span></h3><h3 class="c4"><a name="h.oapjx65rnz4x"></a><span>4&#12289;Prepare medium&#65306;LB liquid medium &nbsp;300ml&#65292;</span></h3><h3 class="c4"><a name="h.oapjx65rnz4x"></a><span>LB medium 150ml </span></h3><h3 class="c4"><a name="h.oapjx65rnz4x"></a><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;TY medium 150ml</span></h3><h3 class="c4"><a name="h.oapjx65rnz4x"></a><span>5&#12289;Prepare SMM medium&#65292;+ pro+ Vb1</span></h3><h1 class="c4"><a name="h.r6ryyomviv4b"></a><span>8.24</span></h1><h3 class="c4"><a name="h.ciewiyq5t66a"></a><span>1&#12289;Prepare SMM medium &nbsp;:MM+pro+Vb1 &nbsp;MM+pro+Vb1+leu</span></h3><h3 class="c4"><a name="h.ciewiyq5t66a"></a><span>2&#12289;check by small gel</span></h3><h1 class="c4"><a name="h.he4e0lgvyqrw"></a><span>8.25</span></h1><h3 class="c4"><a name="h.8kqf064rpjw5"></a><a href="#" name="id.b6b2514dfea5"></a><span>1&#12289;ligate 11-16&amp;18-13 and 5&amp;4 using 3A</span></h3><h3 class="c4"><a name="h.tkxpq18ksh8e"></a><span>Measure density&#65306;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>11-16</span></p></td><td class="c1"><p class="c4"><span>18-13</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>density&#65288;ng/ul&#65289;</span></p></td><td class="c1"><p class="c4"><span>46.5</span></p></td><td class="c1"><p class="c4"><span>136.5</span></p></td><td class="c1"><p class="c4"><span>85.5</span></p></td><td class="c1"><p class="c4"><span>71</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.f8muhyhuv6l2"></a><span>Double enzymes</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>18-3</span></p></td><td class="c1"><p class="c4"><span>11-16</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>DNA</span></p></td><td class="c1"><p class="c4"><span>12</span></p></td><td class="c1"><p class="c4"><span>10</span></p></td><td class="c1"><p class="c4"><span>10</span></p></td><td class="c1"><p class="c4"><span>25</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Buffer4</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>BSA</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td><td class="c1"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>H2O</span></p></td><td class="c1"><p class="c4"><span>29.5</span></p></td><td class="c1"><p class="c4"><span>32.5</span></p></td><td class="c1"><p class="c4"><span>31.5</span></p></td><td class="c1"><p class="c4"><span>16.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>enzyme1</span></p></td><td class="c1"><p class="c4"><span>EcoRI-HF 1.5</span></p></td><td class="c1"><p class="c4"><span>XbaI &nbsp;1</span></p></td><td class="c1"><p class="c4"><span>EcoRI-HF &nbsp;1.5</span></p></td><td class="c1"><p class="c4"><span>XbaI &nbsp;1.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>enzyme 2</span></p></td><td class="c1"><p class="c4"><span>SpeI 1.5</span></p></td><td class="c1"><a href="#" name="id.5ffd2b59663f"></a><p class="c4"><span>PstI &nbsp;1</span></p></td><td class="c1"><p class="c4"><span>SpeI &nbsp;1.5</span></p></td><td class="c1"><p class="c4"><span>PstI &nbsp;1.5</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.ynposbrw4sji"></a><span>Check by gel</span></h3><h3 class="c4"><a name="h.sundqa7jgp4t"></a><span>Ligation of 5&amp;4</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>5</span></p></td><td class="c0"><p class="c4"><span>4</span></p></td><td class="c0"><p class="c4"><span>AC</span></p></td><td class="c0"><p class="c4"><span>T4</span></p></td><td class="c6"><p class="c4"><span>Buffer</span></p></td><td class="c6"><p class="c4"><span>H2O</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>6</span></p></td><td class="c0"><p class="c4"><span>4</span></p></td><td class="c0"><p class="c4"><span>4</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>2</span></p></td><td class="c6"><p class="c4"><span>3</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.ezohoz16dnao"></a><span>Ligation of &nbsp;11-16&amp;18-13&#65306;&#65288;ul&#65289;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>K381001</span></p></td><td class="c0"><p class="c4"><span>18-3</span></p></td><td class="c0"><p class="c4"><span>11-16</span></p></td><td class="c0"><p class="c4"><span>Buffer</span></p></td><td class="c6"><p class="c4"><span>T4</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>5</span></p></td><td class="c0"><p class="c4"><span>6</span></p></td><td class="c0"><p class="c4"><span>6</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.8ylqa0m2w31v"></a><span>2&#12289;inoculate HB101 and BL21 into the solid medium (MM+VB1+Pro and MM+VB1+Pro+Leu)&#65292;culture the Rhizobium etli CFN42 and Sinorhizobium meliloti 1021 on TY solid medium at 30&#8451;</span></h3><h3 class="c29 c4"><a name="h.94y273j4x26h"></a></h3><h1 class="c4"><a name="h.b25s7bsf4qft"></a><span>8.27</span></h1><h3 class="c4"><a name="h.94y273j4x26h"></a><span>1&#12289;transform 18-3&amp;11</span></h3><h3 class="c4"><a name="h.94y273j4x26h"></a><span>2&#12289;pick 3 colonies of 5&amp;4 and culture in l LB liquid medium</span></h3><h3 class="c4"><a name="h.94y273j4x26h"></a><span>3&#12289;culture LeuB and K115001 in LB liquid medium</span></h3><h3 class="c4"><a name="h.94y273j4x26h"></a><span>4&#12289;recover 22,21,3 and ligate 21&amp;3,22&amp;3</span></h3><h3 class="c4"><a name="h.k3ru3lgh65ye"></a><a href="#" name="id.887d11515b76"></a><span>Ligation of 21&amp;3&#65306;(ul)</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c4"><span>22</span></p></td><td class="c1"><p class="c4"><span>3</span></p></td><td class="c1"><p class="c4"><span>T4</span></p></td><td class="c1"><p class="c4"><span>Buffer</span></p></td><td class="c1"><p class="c4"><span>ddH2O</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>7</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>6</span></p></td></tr></tbody></table><h3 class="c29 c4"><a name="h.6pt8yxfqhwg6"></a></h3><h3 class="c4"><a name="h.6pt8yxfqhwg6"></a><span>Ligation of &nbsp;22&amp;3&#65306;(ul)</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c4"><span>21</span></p></td><td class="c1"><p class="c4"><span>3</span></p></td><td class="c1"><p class="c4"><span>T4</span></p></td><td class="c1"><p class="c4"><span>Buffer</span></p></td><td class="c1"><p class="c4"><span>ddH2O</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>8.5</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>4.5</span></p></td></tr></tbody></table><p class="c2"><span></span></p><p class="c2"><span></span></p><h1 class="c4"><a name="h.5bliw8b6jufq"></a><span>8.28</span></h1><h3 class="c4"><a name="h.ee824wmlivl1"></a><span>1&#12289;Prepare medium&#65306;LB liquid medium &nbsp;500ml</span></h3><h3 class="c4"><a name="h.ee824wmlivl1"></a><span>2&#12289;transform 5&amp;4 and 18-3&amp;11-16 again&#65292;and transform leu 21&amp;3 and 22&amp;3</span></h3><h3 class="c4"><a name="h.ee824wmlivl1"></a><span>3&#12289; culture deficient cells</span></h3><h3 class="c4"><a name="h.ee824wmlivl1"></a><span>4&#12289;culture 18-3&amp;11 in LB liquid medium </span></h3><h3 class="c4"><a name="h.ee824wmlivl1"></a><span>8.29</span></h3><h3 class="c4"><a name="h.ee824wmlivl1"></a><span>1&#12289;PCR :5&amp;4 and leu</span></h3><h3 class="c4"><a name="h.awlrj81666pu"></a><span>PCR&#65288;ul&#65289;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c17"><p class="c4"><span>ddH2O</span></p></td><td class="c17"><p class="c4"><span>dNTP</span></p></td><td class="c21"><p class="c4"><span>Buffer</span></p></td><td class="c17"><a href="#" name="id.bd219e189f6f"></a><p class="c4"><span>Primer-F</span></p></td><td class="c21"><p class="c4"><span>Primer-R</span></p></td><td class="c21"><p class="c4"><span>dna</span></p></td><td class="c21"><p class="c4"><span>Taq</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>39.5</span></p></td><td class="c17"><p class="c4"><span>2</span></p></td><td class="c21"><p class="c4"><span>5</span></p></td><td class="c17"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>1</span></p></td><td class="c21"><p class="c4"><span>0.5</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.cbhr3fgm4nrj"></a><span>2&#12289;Presrvation species of 18-3&amp;11 and extract plasmid,measure density, digest</span></h3><h3 class="c4"><a name="h.cbhr3fgm4nrj"></a><span>density&#65288;1&#65289;19.5&#65288;2&#65289;130.5</span></h3><h3 class="c4"><a name="h.cbhr3fgm4nrj"></a><span>digestion</span></h3><h3 class="c4"><a name="h.p91mu85t6yz4"></a><span>single enzyme</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>&#65288;1&#65289;</span></p></td><td class="c9"><p class="c4"><span>&#65288;2&#65289;</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>6</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>BSA</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>2.65</span></p></td><td class="c9"><p class="c4"><span>7.65</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>XbaI</span></p></td><td class="c9"><p class="c4"><span>0.35</span></p></td><td class="c9"><p class="c4"><span>0.35</span></p></td></tr></tbody></table><p class="c4"><span>Double enzymes:</span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>&#65288;1&#65289;</span></p></td><td class="c9"><p class="c4"><span>&#65288;2&#65289;</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>6</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>BSA</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td><td class="c9"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>2.4</span></p></td><td class="c9"><p class="c4"><span>7.4</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>EcoRI</span></p></td><td class="c9"><p class="c4"><span>0.25</span></p></td><td class="c9"><p class="c4"><span>0.25</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>PstI</span></p></td><td class="c9"><p class="c4"><span>0.25</span></p></td><td class="c9"><p class="c4"><span>0.25</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.71fxa3curofb"></a><span>Check by small gel</span></h3><h3 class="c4"><a name="h.71fxa3curofb"></a><span>3&#12289;pick single colony of 5&amp;4&#65292;leu,21&amp;3,22&amp;3&#65292;18-3&amp;11-16 and culture in LB liquid medium&#12290;</span></h3><h3 class="c4"><a name="h.71fxa3curofb"></a><span>4&#12289;prepare TY medium 100ml</span></h3><h3 class="c4"><a name="h.71fxa3curofb"></a><span>8.30</span></h3><h3 class="c4"><a name="h.71fxa3curofb"></a><a href="#" name="id.e846d4e866ce"></a><span>1&#12289;Presrvation species of 5&amp;4,leuB89,leuB13 and extract plasmid,measure density, digest</span></h3><h3 class="c4"><a name="h.71fxa3curofb"></a><span>density&#65306;</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c17"><p class="c4"><span>leuB13&#65288;1&#65289;</span></p></td><td class="c17"><a href="#" name="id.f54c4c23a77f"></a><p class="c4"><span>5&amp;4&#65288;1&#65289;</span></p></td><td class="c21"><p class="c4"><span>5&amp;4&#65288;2&#65289;</span></p></td><td class="c17"><p class="c4"><span>5&amp;4&#65288;3&#65289;</span></p></td><td class="c21"><p class="c4"><span>leuB89&#65288;1&#65289;</span></p></td><td class="c21"><p class="c4"><span>leuB89&#65288;2&#65289;</span></p></td><td class="c21"><p class="c4"><span>leuB89(3)</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>24.5</span></p></td><td class="c17"><p class="c4"><span>188.0</span></p></td><td class="c21"><p class="c4"><span>62.0</span></p></td><td class="c17"><p class="c4"><span>132.0</span></p></td><td class="c21"><p class="c4"><span>6.8</span></p></td><td class="c21"><p class="c4"><span>3.1</span></p></td><td class="c21"><p class="c4"><span>10.8</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.6jtrl26b1tlv"></a><span>Digestion of &nbsp;5&amp;4</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>&#65288;1&#65289;</span></p></td><td class="c13"><p class="c4"><span>&#65288;2&#65289;</span></p></td><td class="c13"><p class="c4"><span>&#65288;3&#65289;</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DNA</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer3</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>ddH2O</span></p></td><td class="c5"><p class="c4"><span>7.5</span></p></td><td class="c13"><p class="c4"><span>6.5</span></p></td><td class="c13"><p class="c4"><span>7.5</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>BSA</span></p></td><td class="c5"><p class="c4"><span>0.1</span></p></td><td class="c13"><p class="c4"><span>0.1</span></p></td><td class="c13"><p class="c4"><span>0.1</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>EcoRI</span></p></td><td class="c5"><p class="c4"><span>0.2</span></p></td><td class="c13"><p class="c4"><span>0.2</span></p></td><td class="c13"><p class="c4"><span>0.2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>PstI</span></p></td><td class="c5"><p class="c4"><span>0.2</span></p></td><td class="c13"><p class="c4"><span>0.2</span></p></td><td class="c13"><p class="c4"><span>0.2</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.9sbx477gdv2a"></a><span>Check by small gel</span></h3><h3 class="c4"><a name="h.9sbx477gdv2a"></a><span>Result:5&amp;4 and leuB successfully ligate</span></h3><h1 class="c4"><a name="h.em1ctnutxs3l"></a><span>8.31</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.3gv9af27ikw"></a><span>1&#12289;did enzyme digestion to 2 part (with psB1AK2 carrier) and K115001 part(with psB1AT3 carrier) and run electrophoresis together with psB1C3. We recycled the carrier part by cutting the gel.</span></h3><h3 class="c4"><a name="h.3gv9af27ikw"></a><span>density&#65306;</span></h3><h3 class="c4"><a name="h.3gv9af27ikw"></a><span>2 part &nbsp; 37.5</span></h3><a href="#" name="id.a5fc2f8264e3"></a><h3 class="c4"><a name="h.3gv9af27ikw"></a><span>K115001 part &nbsp; 120</span></h3><h3 class="c4"><a name="h.p7f0i2i3brxd"></a><span>digestion(ul)</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>K115001</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>9</span></p></td><td class="c9"><p class="c4"><span>9</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>10*H Buffer</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>EcoRI</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>PstI</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>7</span></p></td><td class="c9"><p class="c4"><span>7</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.ox56qgmq045h"></a><span>9.1</span></h1><h3 class="c4"><a name="h.q4spboi6k88h"></a><span>1&#12289;prepare 0147TY medium</span></h3><h3 class="c4"><a name="h.q4spboi6k88h"></a><span>Tryptone &nbsp;5.0g &nbsp;Yeast extract 3.0g &nbsp;CaCl.6H2O &nbsp;1.3g &nbsp;Agar &nbsp;15.0g Distilled water 1.0L</span></h3><h3 class="c4"><a name="h.q4spboi6k88h"></a><span>2&#12289;ligate 18-3&amp;11-16 and 18-3&amp;11</span></h3><h3 class="c4"><a name="h.q4spboi6k88h"></a><span>density&#65306;</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c10"><p class="c4"><span>18-3&#65288;1&#65289;</span></p></td><td class="c10"><p class="c4"><span>18-3&#65288;2&#65289;</span></p></td><td class="c10"><p class="c4"><span>11-16&#65288;1&#65289;</span></p></td><td class="c10"><p class="c4"><span>11-16&#65288;2&#65289;</span></p></td><td class="c10"><p class="c4"><span>K carrier&#65288;1&#65289;</span></p></td><td class="c10"><p class="c4"><span>K carrier&#65288;2&#65289;</span></p></td><td class="c16"><p class="c4"><span>AC&#65288;1&#65289;</span></p></td><td class="c16"><p class="c4"><span>AC(2)</span></p></td></tr><tr class="c12"><td class="c10"><p class="c4"><span>140</span></p></td><td class="c10"><p class="c4"><span>98</span></p></td><td class="c10"><p class="c4"><span>278.5</span></p></td><td class="c10"><p class="c4"><span>203&#65294;0</span></p></td><td class="c10"><p class="c4"><span>17.5</span></p></td><td class="c10"><p class="c4"><span>17.5</span></p></td><td class="c16"><p class="c4"><span>7.9</span></p></td><td class="c16"><p class="c4"><span>13.1</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.hedvpls048g0"></a><span>Digestion:<br></span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>18-3</span></p></td><td class="c13"><p class="c4"><span>11</span></p></td><td class="c13"><p class="c4"><span>11-16</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DNA</span></p></td><td class="c5"><p class="c4"><span>7</span></p></td><td class="c13"><p class="c4"><span>10</span></p></td><td class="c13"><p class="c4"><span>4</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer</span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>enzyme1</span></p></td><td class="c5"><p class="c4"><span>E &nbsp;1</span></p></td><td class="c13"><p class="c4"><span>X &nbsp; 1</span></p></td><td class="c13"><p class="c4"><span>X &nbsp;1</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>enzyme 2</span></p></td><td class="c5"><p class="c4"><span>S &nbsp;1</span></p></td><td class="c13"><p class="c4"><span>P &nbsp; &nbsp;1</span></p></td><td class="c13"><p class="c4"><span>P &nbsp; 1</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DdH2O</span></p></td><td class="c5"><p class="c4"><span>9</span></p></td><td class="c13"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>12</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.3yfownuunxbz"></a><span>Ligation:</span></h3><h3 class="c4"><a name="h.3yfownuunxbz"></a><span>18-3&#65306;3ul &nbsp;16:1ul &nbsp; 2&#65306;6ul &nbsp;T4&#65306;1ul &nbsp;Buffer&#65306;2ul &nbsp; H2O&#65306;7ul</span></h3><h3 class="c29 c4"><a name="h.3yfownuunxbz"></a></h3><h3 class="c4"><a name="h.3yfownuunxbz"></a><span>18-3&#65306;3ul &nbsp;11-16&#65306;4ul &nbsp;2&#65306;6ul &nbsp; T4&#65306;1ul &nbsp; Buffer&#65306;2ul &nbsp;ddH2O&#65306;4ul</span></h3><h1 class="c4"><a name="h.tmngci922iwu"></a><span>9.2 </span></h1><h3 class="c4"><a name="h.wkppfbc5lyza"></a><span>1&#12289;Presrvation species of leuB and extract plasmid,measure density, digest</span></h3><h3 class="c4"><a name="h.wkppfbc5lyza"></a><span>density&#65306;</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><h3 class="c4"><a name="h.wkppfbc5lyza"></a><span>1</span></h3></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>3</span></p></td><td class="c13"><p class="c4"><span>4</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>292.5</span></p></td><td class="c5"><p class="c4"><span>21.5</span></p></td><td class="c13"><p class="c4"><span>223.5</span></p></td><td class="c13"><p class="c4"><span>32.0</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.bi0spzoaltti"></a><span>Digestion:</span></h1><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>leuB</span></p></td><td class="c9"><a href="#" name="id.d705116cf752"></a><p class="c4"><span>K(pSB1C3)</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>EcoRI</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>PstI</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>14</span></p></td><td class="c9"><p class="c4"><span>8.5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>7.5</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.9sfoto1zfgis"></a><span>Check by small gel:</span></h3><p class="c2"><span></span></p><h3 class="c4"><a name="h.bjhs1az2y57t"></a><span>recover&#65306;</span></h3><h3 class="c4"><a name="h.bjhs1az2y57t"></a><span>2&#12289;pick single colony of 18-3&amp;11-16 and 18-3&amp;11 and culture in shaker</span></h3><h3 class="c4"><a name="h.bjhs1az2y57t"></a><span>3&#12289;transform BBa_I751250,as LuxI_AHL producer&#12290;Transform BBa_J04450(placI+mRFP)</span></h3><h3 class="c4"><a name="h.bjhs1az2y57t"></a><span>9.3</span></h3><h3 class="c4"><a name="h.bjhs1az2y57t"></a><span>1&#12289;ligate leuB, K(pSB1C3) carrier and transform</span></h3><h3 class="c4"><a name="h.bjhs1az2y57t"></a><span>density&#65306;</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>Before Concentration</span></p></td><td class="c9"><p class="c4"><span>After &nbsp;Concentration</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>LeuB</span></p></td><td class="c9"><p class="c4"><span>15</span></p></td><td class="c9"><p class="c4"><span>36</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>pSB1C3</span></p></td><td class="c9"><p class="c4"><span>8</span></p></td><td class="c9"><p class="c4"><span>25</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.6ii9ncjvqxui"></a><span>ligation</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c4"><span>LeuB</span></p></td><td class="c1"><p class="c4"><span>Psb1c3</span></p></td><td class="c1"><p class="c4"><span>T4</span></p></td><td class="c1"><p class="c4"><span>Buffer</span></p></td><td class="c1"><p class="c4"><span>DdH2O</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>7</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.im43403pxmes"></a><span>2&#12289;extract plasmid of 8-3&amp;11-16 and 18-3&amp;11, digest&#65292;and check by small gel</span></h3><h3 class="c4"><a name="h.im43403pxmes"></a><span>Density:</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c34"><p class="c4"><span>18-3&amp;11-161</span></p></td><td class="c34"><p class="c4"><span>18-3&amp;11-162</span></p></td><td class="c18"><p class="c4"><span>18-3&amp;11 1</span></p></td><td class="c18"><a href="#" name="id.ad39c9b90daa"></a><p class="c4"><span>18-3&amp;11 2</span></p></td><td class="c52"><p class="c4"><span>5-4&#65288;AC&#65289;1 </span></p></td><td class="c56"><p class="c4"><span>5-4&#65288;AC&#65289; 2</span></p></td><td class="c53"><p class="c4"><span>2-6-3</span></p></td></tr><tr class="c12"><td class="c34"><p class="c4"><span>265</span></p></td><td class="c34"><p class="c4"><span>242</span></p></td><td class="c18"><p class="c4"><span>192</span></p></td><td class="c18"><p class="c4"><span>217.5</span></p></td><td class="c52"><p class="c4"><span>193</span></p></td><td class="c56"><p class="c4"><span>72</span></p></td><td class="c53"><p class="c4"><span>63.5</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.qevj1yc7kkwj"></a><span>digestion&#65306;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>18-3&amp;11-16(1)</span></p></td><td class="c1"><p class="c4"><span>18-3&amp;11-16(2)</span></p></td><td class="c1"><p class="c4"><span>18-3&amp;11(1)</span></p></td><td class="c1"><p class="c4"><span>18-3&amp;11(2)</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>DNA</span></p></td><td class="c1"><p class="c4"><span>3.5</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>5</span></p></td><td class="c1"><p class="c4"><span>4.5</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>Buffer</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>EcoRI</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>PstI</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>ddH2O</span></p></td><td class="c1"><p class="c4"><span>12.5</span></p></td><td class="c1"><p class="c4"><span>12</span></p></td><td class="c1"><p class="c4"><span>11</span></p></td><td class="c1"><p class="c4"><span>11.5</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.s7dxsac1fmjh"></a><span>Check by gel&#65306;</span></h3><p class="c2"><span></span></p><h3 class="c4"><a name="h.f5jqe09y4j66"></a><span>2&#12289;ligate 5-4&amp;2-6-3</span></h3><h3 class="c4"><a name="h.6o1u60ekkzdx"></a><span>ligation</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>K</span></p></td><td class="c0"><p class="c4"><span>5-4</span></p></td><td class="c0"><p class="c4"><span>2-6-3</span></p></td><td class="c0"><p class="c4"><span>T4&#36830;&#25509;&#37238;</span></p></td><td class="c6"><p class="c4"><span>Buffer</span></p></td><td class="c6"><p class="c4"><span>DdH2O</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>6</span></p></td><td class="c0"><p class="c4"><span>4</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>2</span></p></td><td class="c6"><p class="c4"><span>5</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.h3yoy287vb0g"></a><span>9&#65294;4</span></h1><h3 class="c4"><a name="h.djojukljacfp"></a><span>1&#12289;transform 5-4&amp;2-6-3&#65292;extract plasmid of &nbsp;2-6-3&#19982;2</span></h3><h3 class="c4"><a name="h.djojukljacfp"></a><span>2&#12289;transform Pea Rhizobium</span></h3><h3 class="c4"><a name="h.djojukljacfp"></a><span>9.5</span></h3><h3 class="c4"><a name="h.djojukljacfp"></a><span>1&#12289;Presrvation species of leuB and extract plasmid,measure density, digest, PCR ,check by gel</span></h3><h3 class="c4"><a name="h.djojukljacfp"></a><span>PCR</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>2</span></p></td><td class="c6"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>ddH2O</span></p></td><td class="c0"><p class="c4"><span>37.5</span></p></td><td class="c0"><p class="c4"><span>37</span></p></td><td class="c0"><p class="c4"><span>36.5</span></p></td><td class="c6"><p class="c4"><span>36.5</span></p></td><td class="c6"><p class="c4"><span>36.5</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>dNTPmix</span></p></td><td class="c0"><p class="c4"><span>4</span></p></td><td class="c0"><p class="c4"><span>4</span></p></td><td class="c0"><p class="c4"><span>4</span></p></td><td class="c6"><p class="c4"><span>4</span></p></td><td class="c6"><p class="c4"><span>4</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>Buffer</span></p></td><td class="c0"><p class="c4"><span>5</span></p></td><td class="c0"><p class="c4"><span>5</span></p></td><td class="c0"><p class="c4"><span>5</span></p></td><td class="c6"><p class="c4"><span>5</span></p></td><td class="c6"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>VF2</span></p></td><td class="c0"><p class="c4"><span>1.5</span></p></td><td class="c0"><p class="c4"><span>1.5</span></p></td><td class="c0"><p class="c4"><span>1.5</span></p></td><td class="c6"><p class="c4"><span>1.5</span></p></td><td class="c6"><p class="c4"><span>1.5</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>VR</span></p></td><td class="c0"><p class="c4"><span>1.5</span></p></td><td class="c0"><p class="c4"><span>1.5</span></p></td><td class="c0"><p class="c4"><span>1.5</span></p></td><td class="c6"><p class="c4"><span>1.5</span></p></td><td class="c6"><p class="c4"><span>1.5</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>tamplate</span></p></td><td class="c0"><p class="c4"><span>0</span></p></td><td class="c0"><p class="c4"><span>0.5</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>Taq</span></p></td><td class="c0"><p class="c4"><span>0.5</span></p></td><td class="c0"><p class="c4"><span>0.5</span></p></td><td class="c0"><p class="c4"><span>0.5</span></p></td><td class="c6"><p class="c4"><span>0.5</span></p></td><td class="c6"><p class="c4"><span>0.5</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.4vezb87sj3gs"></a><span>Check by gel </span></h3><p class="c4"><img height="527" src="images/image02.jpg" width="527"></p><h3 class="c4"><a name="h.72c4zg6pthgu"></a><span>digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span>LeuB &nbsp;1</span></p></td><td class="c9"><p class="c4"><span>LeuB &nbsp;3</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA</span></p></td><td class="c9"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>BufferM</span></p></td><td class="c9"><p class="c4"><span>0.5</span></p></td><td class="c9"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O</span></p></td><td class="c9"><p class="c4"><span>1.1</span></p></td><td class="c9"><p class="c4"><span>1.1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>SpeI</span></p></td><td class="c9"><p class="c4"><span>0.2</span></p></td><td class="c9"><p class="c4"><span>0.2</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>XbaI</span></p></td><td class="c9"><p class="c4"><span>0.2</span></p></td><td class="c9"><p class="c4"><span>0.2</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.prk8j67a3erf"></a><span>Check by gel </span></h3><h1 class="c4"><a name="h.ftq4x9fezl9p"></a><span>9.6</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.tzu0yabpanct"></a><span>1&#12289;Presrvation species of 54263 and extract plasmid, digest, PCR ,check by gel</span></h3><h3 class="c29 c4"><a name="h.tzu0yabpanct"></a></h3><h3 class="c4"><a name="h.3i6aolmrnp3z"></a><span>digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DNA</span></p></td><td class="c5"><p class="c4"><span>1.4</span></p></td><td class="c13"><p class="c4"><span>1.4</span></p></td><td class="c13"><p class="c4"><span>1.7</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer</span></p></td><td class="c5"><p class="c4"><span>0.5</span></p></td><td class="c13"><p class="c4"><span>0.5</span></p></td><td class="c13"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DdH2O</span></p></td><td class="c5"><p class="c4"><span>2.5</span></p></td><td class="c13"><p class="c4"><span>2.5</span></p></td><td class="c13"><p class="c4"><span>2.2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>EcoRI</span></p></td><td class="c5"><p class="c4"><span>0.3</span></p></td><td class="c13"><p class="c4"><span>0.3</span></p></td><td class="c13"><p class="c4"><span>0.3</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>PstI</span></p></td><td class="c5"><p class="c4"><span>0.3</span></p></td><td class="c13"><p class="c4"><span>0.3</span></p></td><td class="c13"><p class="c4"><span>0.3</span></p></td></tr></tbody></table><h1 class="c49 c4"><a name="h.woe6zwxsksvp"></a></h1><h1 class="c4"><a name="h.7wkxrcs7xpbv"></a><span>9.7</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.uxiz6xa9xofa"></a><span>1&#12289;measure OD of Deficient cell</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c4"><span>MM&#65288;leuB-&#65289;</span></p></td><td class="c0"><p class="c4"><span>1 &nbsp;leuB+</span></p></td><td class="c0"><p class="c4"><span>2 &nbsp; leuB+</span></p></td><td class="c6"><p class="c4"><span>3 &nbsp; leuB+</span></p></td><td class="c6"><p class="c4"><span>4 &nbsp;leuB+</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>OD&#65288;A340&#65289;</span></p></td><td class="c0"><p class="c4"><span>1.611-1</span></p></td><td class="c0"><p class="c4"><span>1.698-1</span></p></td><td class="c0"><p class="c4"><span>1.818-1</span></p></td><td class="c6"><p class="c4"><span>1.548-1</span></p></td><td class="c6"><p class="c4"><span>1.881-1</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.w97sjfemswol"></a><span>2&#12289;digest &nbsp;leuB&#12289;&#9314;&#12289;K115001</span></h3><h3 class="c4"><a name="h.mfs7a181f07r"></a><span>digestion&#65306;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c26"><p class="c2"><span></span></p></td><td class="c30"><p class="c4"><span>8</span></p></td><td class="c25"><p class="c4"><span>6</span></p></td><td class="c46"><p class="c4"><span>LeuB</span></p></td><td class="c30"><p class="c4"><span>3</span></p></td><td class="c24"><p class="c4"><span>T1</span></p></td><td class="c26"><p class="c4"><span>T2</span></p></td><td class="c24"><p class="c4"><span>T3</span></p></td></tr><tr class="c12"><td class="c26"><p class="c4"><span>DNA</span></p></td><td class="c30"><p class="c4"><span>9</span></p></td><td class="c25"><p class="c4"><span>9</span></p></td><td class="c46"><p class="c4"><span>10</span></p></td><td class="c30"><p class="c4"><span>10</span></p></td><td class="c24"><p class="c4"><span>10</span></p></td><td class="c26"><p class="c4"><span>13</span></p></td><td class="c24"><p class="c4"><span>12</span></p></td></tr><tr class="c12"><td class="c26"><p class="c4"><span>Buffer</span></p></td><td class="c30"><p class="c4"><span>2</span></p></td><td class="c25"><p class="c4"><span>2</span></p></td><td class="c46"><p class="c4"><span>2</span></p></td><td class="c30"><p class="c4"><span>2</span></p></td><td class="c24"><p class="c4"><span>2</span></p></td><td class="c26"><p class="c4"><span>2</span></p></td><td class="c24"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c26"><p class="c4"><span>DdH2O</span></p></td><td class="c30"><p class="c4"><span>7</span></p></td><td class="c25"><p class="c4"><span>6.6</span></p></td><td class="c46"><p class="c4"><span>5.6</span></p></td><td class="c30"><p class="c4"><span>5.6</span></p></td><td class="c24"><p class="c4"><span>5.6</span></p></td><td class="c26"><p class="c4"><span>2.6</span></p></td><td class="c24"><p class="c4"><span>3.6</span></p></td></tr><tr class="c12"><td class="c26"><p class="c4"><span>EcoRI</span></p></td><td class="c30"><p class="c4"><span>1</span></p></td><td class="c25"><p class="c4"><span>1.2</span></p></td><td class="c46"><p class="c4"><span>1.2</span></p></td><td class="c30"><p class="c4"><span>1.2</span></p></td><td class="c24"><p class="c4"><span>1.2</span></p></td><td class="c26"><p class="c4"><span>1.2</span></p></td><td class="c24"><p class="c4"><span>1.2</span></p></td></tr><tr class="c12"><td class="c26"><p class="c4"><span>SpeI</span></p></td><td class="c30"><p class="c4"><span>1</span></p></td><td class="c25"><p class="c4"><span>1.2</span></p></td><td class="c46"><p class="c4"><span>1.2</span></p></td><td class="c30"><p class="c4"><span>1.2</span></p></td><td class="c24"><p class="c4"><span>1.2</span></p></td><td class="c26"><p class="c4"><span>1.2</span></p></td><td class="c24"><p class="c4"><span>1.2</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.5mlvofbvkc4i"></a><span>density&#65306;</span></h3><h3 class="c4"><a name="h.5mlvofbvkc4i"></a><span>LeuB 98.0</span></h3><h3 class="c4"><a name="h.5mlvofbvkc4i"></a><span>T &nbsp;1:93.5 &nbsp; &nbsp; 2:73.5 &nbsp; &nbsp;3:87</span></h3><h3 class="c4"><a name="h.5mlvofbvkc4i"></a><span>3 &nbsp;1:97 &nbsp; &nbsp; &nbsp; 2:73 &nbsp; &nbsp; 3:82</span></h3><h3 class="c4"><a name="h.5mlvofbvkc4i"></a><span>9.8</span></h3><h3 class="c4"><a name="h.5mlvofbvkc4i"></a><span>1&#12289;extract plasmid of 6&#65292;and digest with LeuB</span></h3><h3 class="c4"><a name="h.9dqcvm6xpq2y"></a><span>Density of 6&#65306;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>57</span></p></td><td class="c9"><p class="c4"><span>68</span></p></td><td class="c9"><p class="c4"><span>50</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.ufa9zdoc4n5v"></a><span>digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>LeuB</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DNA</span></p></td><td class="c5"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>10</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer</span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DdH2O</span></p></td><td class="c5"><p class="c4"><span>9.6</span></p></td><td class="c13"><p class="c4"><span>9.6</span></p></td><td class="c13"><p class="c4"><span>5.6</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>EcoRI</span></p></td><td class="c5"><p class="c4"><span>1.2</span></p></td><td class="c13"><p class="c4"><span>1.2</span></p></td><td class="c13"><p class="c4"><span>1.2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>SpeI</span></p></td><td class="c5"><p class="c4"><span>1.2</span></p></td><td class="c13"><p class="c4"><span>1.2</span></p></td><td class="c13"><p class="c4"><span>1.2</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.tgspjxa7cy05"></a><span>2&#12289;ligate leuB&amp;3</span></h3><h3 class="c4"><a name="h.tgspjxa7cy05"></a><span>ligation&#65306;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>T4</span></p></td><td class="c0"><p class="c4"><span>Buffer</span></p></td><td class="c0"><p class="c4"><span>leuB</span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c6"><p class="c4"><span>T carrier</span></p></td><td class="c6"><p class="c4"><span>DdH2O</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c0"><p class="c4"><span>0.7</span></p></td><td class="c6"><p class="c4"><span>10</span></p></td><td class="c6"><p class="c4"><span>3.3</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.2tn7lcrp7780"></a><span>9.9</span></h1><h3 class="c4"><a name="h.ix5rjouwfyue"></a><span>1&#12289;check 6, leuB, 8</span></h3><h3 class="c4"><a name="h.ix5rjouwfyue"></a><span>Gel&#65306;</span></h3><h3 class="c4"><a name="h.ix5rjouwfyue"></a><span>Ligate 8&amp;6&#12289;6&amp;leuB</span></h3><h3 class="c4"><a name="h.jizjvyn0z6vd"></a><span>Ligation of 8&amp;6&#65306;</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>T carrier</span></p></td><td class="c0"><p class="c4"><span>8</span></p></td><td class="c0"><p class="c4"><span>6</span></p></td><td class="c0"><p class="c4"><span>T4</span></p></td><td class="c6"><p class="c4"><span>Buffer</span></p></td><td class="c6"><p class="c4"><span>DdH2O</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>10</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>2</span></p></td><td class="c6"><p class="c4"><span>5</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.sojo5w7w5q9c"></a><span>Ligation of &nbsp;6&amp;leuB</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>T carrier</span></p></td><td class="c0"><p class="c4"><span>6</span></p></td><td class="c0"><p class="c4"><span>LeuB</span></p></td><td class="c0"><p class="c4"><span>T4</span></p></td><td class="c6"><p class="c4"><span>Buffer</span></p></td><td class="c6"><p class="c4"><span>DdH2O</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>10</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>2</span></p></td><td class="c6"><p class="c4"><span>3</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.vnbfx386r4a2"></a><span>9.10</span></h1><h3 class="c4"><a name="h.90ycqjb04vb7"></a><span>1&#12289;transform 8&amp;6 and &nbsp;leuB</span></h3><h3 class="c4"><a name="h.90ycqjb04vb7"></a><span>2&#12289;culture leuB&amp;3 in LB medium</span></h3><h3 class="c4"><a name="h.90ycqjb04vb7"></a><span>3&#12289;device checking&#65306;</span></h3><h3 class="c4"><a name="h.90ycqjb04vb7"></a><span>Device1 has strong Fluorescent and device 2 hasslight one</span></h3><h3 class="c4"><a name="h.90ycqjb04vb7"></a><span>Picture:</span></h3><h1 class="c4 c49"><a name="h.1ummjyzat1wc"></a></h1><h1 class="c4"><a name="h.p1bniw9xi6lt"></a><span>9.11</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.j1mlghz0dpg9"></a><span>1&#12289;PCR&#65306;sinR&#12289;pro-RBS-sinR&#12289;raiR&#12289;raiR-pro</span></h3><p class="c2"><span></span></p><h3 class="c4"><a name="h.fcdrc1dlv06o"></a><span>density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c37"><p class="c4"><span>sinI</span></p></td><td class="c37"><p class="c4"><span>12.8</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>SinI pro</span></p></td><td class="c37"><p class="c4"><span>9.6</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>Pro-RBS-sinI</span></p></td><td class="c37"><p class="c4"><span>15.1</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>RaiI</span></p></td><td class="c37"><p class="c4"><span>14.2</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>RaiI pro</span></p></td><td class="c37"><p class="c4"><span>9.4</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>Pro-RBS-raiI</span></p></td><td class="c37"><p class="c4"><span>11.4</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>SinR pro</span></p></td><td class="c37"><p class="c4"><span>11.5</span></p></td></tr><tr class="c12"><td class="c37"><p class="c4"><span>Pro-RBS-raiR</span></p></td><td class="c37"><p class="c4"><span>15.0</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.ko2zysom0iot"></a><span>2&#12289;pick single colony of 6&amp;leuB</span></h3><h3 class="c4"><a name="h.ko2zysom0iot"></a><span>9.12</span></h3><h3 class="c4"><a name="h.ko2zysom0iot"></a><span>1&#12289;digest 21&#12289;22 and ligate</span></h3><h3 class="c4"><a name="h.ko2zysom0iot"></a><span>digestion</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>21</span></p></td><td class="c13"><p class="c4"><span>22</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer</span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>NDA</span></p></td><td class="c5"><p class="c4"><span>13.6</span></p></td><td class="c13"><p class="c4"><span>15</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DdH2O</span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>0.6</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>EcoRI</span></p></td><td class="c5"><p class="c4"><span>1.2</span></p></td><td class="c13"><p class="c4"><span>1.2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>SpeI</span></p></td><td class="c5"><p class="c4"><span>1.2</span></p></td><td class="c13"><p class="c4"><span>1.2</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.7q3di5col2wi"></a><span>ligation</span></h3><h3 class="c4"><a name="h.7q3di5col2wi"></a><span>21&amp;3</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>21</span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c0"><p class="c4"><span>Buffer </span></p></td><td class="c0"><p class="c4"><span>T4</span></p></td><td class="c6"><p class="c4"><span>DdH2O</span></p></td><td class="c6"><p class="c4"><span>T</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>3</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>3</span></p></td><td class="c6"><p class="c4"><span>10</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.8g2gy677wg7m"></a><span>22&amp;3</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>22</span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c0"><p class="c4"><span>Buffer</span></p></td><td class="c0"><p class="c4"><span>T4</span></p></td><td class="c6"><p class="c4"><span>DdH2O</span></p></td><td class="c6"><p class="c4"><span>T</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>5</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>10</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.78fosknlspxo"></a><span>2&#12289;extract plasmid of leuB-6 and measure density</span></h3><h3 class="c4"><a name="h.78fosknlspxo"></a><span>density&#65306;16.5</span></h3><h1 class="c4"><a name="h.z2gmdjiy1hon"></a><span>9&#65294;13</span></h1><h3 class="c4"><a name="h.slxf8ucbmfw5"></a><span>1&#12289;transform and culture 21&amp;3,22&amp;3,8&amp;6&#65292;recover PSB1C3 carrier</span></h3><h3 class="c4"><a name="h.xnxdtzar9e2f"></a><span>2.Pcr</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c17"><p class="c4"><span>ddH2O</span></p></td><td class="c17"><p class="c4"><span>Pfu with MgSO4 buffer</span></p></td><td class="c21"><p class="c4"><span>dNTP</span></p></td><td class="c17"><p class="c4"><span>PF</span></p></td><td class="c21"><p class="c4"><span>PR</span></p></td><td class="c21"><p class="c4"><span>Template</span></p></td><td class="c21"><p class="c4"><span>Pfu</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>38</span><span class="c3">&micro;</span><span>l</span></p></td><td class="c17"><p class="c4"><span>5</span><span class="c3">&micro;</span><span>l</span></p></td><td class="c21"><p class="c4"><span>4</span><span class="c3">&micro;</span><span>l</span></p></td><td class="c17"><p class="c4"><span>1</span><span class="c3">&micro;</span><span>l</span></p></td><td class="c21"><p class="c4"><span>1</span><span class="c3">&micro;</span><span>l</span></p></td><td class="c21"><p class="c4"><span>0.5</span><span class="c3">&micro;</span><span>l</span></p></td><td class="c21"><p class="c4"><span>0.5</span><span class="c3">&micro;</span><span>l</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.vyykrtrhy0we"></a><span>Results&#65306;failed</span></h3><h3 class="c4"><a name="h.vyykrtrhy0we"></a><span>3.digest sinR, sinI,pro- sinI</span></h3><h3 class="c4"><a name="h.2kxssry4fo1d"></a><span>Digestion:20</span><span class="c3">&micro;</span><span>l</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c5"><p class="c4"><span>sinR</span></p></td><td class="c13"><p class="c4"><span>sinI</span></p></td><td class="c13"><p class="c4"><span>pro- sinI</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DNA</span></p></td><td class="c5"><p class="c4"><span>10</span></p></td><td class="c13"><p class="c4"><span>11</span></p></td><td class="c13"><p class="c4"><span>16</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer 10</span><span class="c3">&times;</span><span>H</span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>ddH2O</span></p></td><td class="c5"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>5</span></p></td><td class="c13"><p class="c4"><span>0</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>EcoRI</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>PstI</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.3n89uor9jv7a"></a><span>9.15</span></h1><h3 class="c4"><a name="h.mwd71ak11zm"></a><span>Digestion of Leu&amp;3 :20</span><span class="c3">&micro;</span><span>l</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c4"><span>number</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DNA(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>10</span></p></td><td class="c13"><p class="c4"><span>10</span></p></td><td class="c13"><p class="c4"><span>9</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer 10</span><span class="c3">&times;</span><span>H(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>ddH2O(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>6</span></p></td><td class="c13"><p class="c4"><span>7</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span class="c7">Xba</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.s3eqhw38nr24"></a><span>9.16</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.8um0wy4nrt8u"></a><span>Digest 6 and leub</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span>6</span></p></td><td class="c9"><p class="c4"><span>Leu</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>DNA(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c9"><p class="c4"><span>15</span></p></td><td class="c9"><p class="c4"><span>9</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Buffer 10</span><span class="c3">&times;</span><span>M(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>ddH2O(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>7</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>enzymew</span></p></td><td class="c9"><p class="c4"><span class="c7">Spe</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l) </span><span class="c3">1</span></p></td><td class="c9"><p class="c4"><span class="c7">Xba</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l) 1</span></p></td></tr><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c2"><span></span></p></td><td class="c9"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l) 1</span></p></td></tr></tbody></table><h1 class="c4"><a name="h.5jlkua87etap"></a><span>9.18</span></h1><h3 class="c4"><a name="h.wll2rnwql4h5"></a><span>Ligate Leu and 6 using 3A</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c2"><span></span></p></td><td class="c1"><p class="c4"><span>Leu</span></p></td><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>buffer</span></p></td><td class="c1"><p class="c4"><span>T4</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>10</span><span class="c3">&micro;</span><span>l</span></p></td><td class="c1"><p class="c4"><span class="c3">2&micro;</span><span>l</span></p></td><td class="c1"><p class="c4"><span class="c3">6&micro;</span><span>l</span></p></td><td class="c1"><p class="c4"><span class="c3">2&micro;</span><span>l</span></p></td><td class="c1"><p class="c4"><span class="c3">1&micro;</span><span>l</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.8d09k2ryw264"></a><span>Gel picture&#65306;</span></h3><p class="c4"><img height="273" src="images/image03.jpg" width="562"></p><h3 class="c4"><a name="h.pzff1yr1mq6n"></a><span>Digest 6</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c4"><span>number</span></p></td><td class="c1"><p class="c4"><span>DNA(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c1"><p class="c4"><span>Buffer 10</span><span class="c3">&times;</span><span>&nbsp;(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c1"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c1"><p class="c4"><span>ddH2O(</span><span class="c3">&micro;</span><span>l)</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>9</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>8</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.rjhja6hena4r"></a><span>9.20</span></h1><p class="c2"><span></span></p><h3 class="c4"><a name="h.doclcz4jf63n"></a><span>Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c38"><p class="c4"><span>number</span></p></td><td class="c50"><p class="c4"><span class="c27 c3">n</span><span class="c27 c7">g/</span><span class="c27 c3">&micro;</span><span class="c27 c7">l</span></p></td><td class="c50"><p class="c4"><span class="c7">A260</span><span class="c3">&#8725;</span><span class="c7">A280</span></p></td></tr><tr class="c12"><td class="c38"><p class="c4"><span>6</span></p></td><td class="c50"><p class="c4"><span>179.5</span></p></td><td class="c50"><p class="c4"><span>1.851</span></p></td></tr><tr class="c12"><td class="c38"><p class="c4"><span>pSB1C3-1</span></p></td><td class="c50"><p class="c4"><span>6.3</span></p></td><td class="c50"><p class="c2"><span></span></p></td></tr><tr class="c12"><td class="c38"><p class="c4"><span>pSB1C3-2</span></p></td><td class="c50"><p class="c4"><span>8.6</span></p></td><td class="c50"><p class="c4"><span>3.510</span></p></td></tr><tr class="c12"><td class="c38"><p class="c4"><span>pSB1C3-3</span></p></td><td class="c50"><p class="c4"><span>5.1</span></p></td><td class="c50"><p class="c2"><span></span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.xbnykbw91qca"></a><span>Digest Leu</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c4"><span>DNA(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c1"><p class="c4"><span>Buffer 10</span><span class="c3">&times;</span><span>M(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c1"><p class="c19 c4"><span>ddH2O(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c1"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c1"><p class="c4"><span class="c7">Xba</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>8</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.ebm9jwpwvzye"></a><span>Ligate Leu&amp; 6</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c1"><p class="c4"><span>Leu</span></p></td><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>buffer</span></p></td><td class="c1"><p class="c4"><span>T4</span></p></td><td class="c1"><p class="c4"><span>ddH2O</span></p></td></tr><tr class="c12"><td class="c1"><p class="c4"><span>6</span></p></td><td class="c1"><p class="c4"><span>4</span></p></td><td class="c1"><p class="c4"><span>2</span></p></td><td class="c1"><p class="c4"><span>1</span></p></td><td class="c1"><p class="c4"><span>7</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.gx5ptw3fqhxn"></a><span>9.21</span></h1><h3 class="c4"><a name="h.t4pz3r81wrf0"></a><span>Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span class="c27 c3">n</span><span class="c27 c7">g/</span><span class="c27 c3">&micro;</span><span class="c27 c7">l</span></p></td><td class="c9"><p class="c4"><span class="c7">A260</span><span class="c3">&#8725;</span><span class="c7">A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>rai</span><span class="c3">&#8544;</span><span>pro-3</span></p></td><td class="c9"><p class="c4"><span>29.5</span></p></td><td class="c9"><p class="c4"><span>1.666</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>rai</span><span class="c3">&#8544;</span><span>pro-2</span></p></td><td class="c9"><p class="c4"><span>28.5</span></p></td><td class="c9"><p class="c4"><span>1.885</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>rai</span><span class="c3">&#8544;</span><span>pro-1</span></p></td><td class="c9"><p class="c4"><span>23.0</span></p></td><td class="c9"><p class="c4"><span>1.818</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>C0076</span></p></td><td class="c9"><p class="c4"><span>161.0</span></p></td><td class="c9"><p class="c4"><span>1.793</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>E0020e0fp-1</span></p></td><td class="c9"><p class="c4"><span>28.5</span></p></td><td class="c9"><p class="c4"><span>1.676</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>E0020e0fp-2</span></p></td><td class="c9"><p class="c4"><span>19.5</span></p></td><td class="c9"><p class="c4"><span>2.191</span></p></td></tr></tbody></table><h3 class="c29 c4"><a name="h.ahkncaylcg39"></a></h3><h3 class="c4"><a name="h.wwp8s7phrpou"></a><span>Digestion 5</span><span class="c3">&micro;</span><span>l</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c4"><span>number</span></p></td><td class="c5"><p class="c4"><span>rai</span><span class="c3">&#8544;</span><span>pro-1</span></p></td><td class="c13"><p class="c4"><span>rai</span><span class="c3">&#8544;</span><span>pro-2</span></p></td><td class="c13"><p class="c4"><span>rai</span><span class="c3">&#8544;</span><span>pro-3</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>DNA(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>4</span></p></td><td class="c13"><p class="c4"><span>4</span></p></td><td class="c13"><p class="c4"><span>4</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>Buffer 10</span><span class="c3">&times;</span><span>H (</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>0.5</span></p></td><td class="c13"><p class="c4"><span>0.5</span></p></td><td class="c13"><p class="c4"><span>0.5</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>EcoRI(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>0.25</span></p></td><td class="c13"><p class="c4"><span>0.25</span></p></td><td class="c13"><p class="c4"><span>0.25</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>PstI(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c5"><p class="c4"><span>0.25</span></p></td><td class="c13"><p class="c4"><span>0.25</span></p></td><td class="c13"><p class="c4"><span>0.25</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.5joce4249z7i"></a><span>20</span><span class="c3">&micro;</span><span>l</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>number</span></p></td><td class="c0"><p class="c4"><span>DNA(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c0"><p class="c4"><span>Buffer 10</span><span class="c3">&times;</span><span>M(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c0"><p class="c4 c19"><span>ddH2O(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c6"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c6"><p class="c4"><span class="c7">Xba</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>C0076</span></p></td><td class="c0"><p class="c4"><span>6.5</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c0"><p class="c4"><span>9.5</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td><td class="c6"><p class="c4"><span>1</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.c6a30hxeop1y"></a><span>50</span><span class="c3">&micro;</span><span>l</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>number</span></p></td><td class="c0"><p class="c4"><span>DNA(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c0"><p class="c4"><span>Buffer 10</span><span class="c3">&times;</span><span>M(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c0"><p class="c19 c4"><span>ddH2O(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c6"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c6"><p class="c4"><span class="c7">Xba</span><span class="c3">&#8544;</span><span>(</span><span class="c3">&micro;</span><span>l)</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>E0020</span></p></td><td class="c0"><p class="c4"><span>24</span></p></td><td class="c0"><p class="c4"><span>5</span></p></td><td class="c0"><p class="c4"><span>18</span></p></td><td class="c6"><p class="c4"><span>1.5</span></p></td><td class="c6"><p class="c4"><span>1.5</span></p></td></tr></tbody></table><p class="c2"><span></span></p><p class="c2"><span></span></p><p class="c2"><span></span></p><h1 class="c4"><a name="h.dx3lcv3d11b7"></a><span>9.23</span></h1><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c17"><p class="c4"><span>devive</span></p></td><td class="c23"><p class="c4 c22"><span>&#19968;</span></p></td><td class="c14"><p><span></span></p></td><td class="c43"><p class="c4 c22"><span>&#20108;</span></p></td><td class="c17"><p><span></span></p></td><td class="c17"><p class="c4 c22"><span>&#19977;</span></p></td><td class="c23"><p><span></span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>number</span></p></td><td class="c23"><p class="c4"><span>5-4(AC)</span></p></td><td class="c14"><p class="c4"><span>Leu-6(A)</span></p></td><td class="c43"><p class="c4"><span>18-3-11(AK)</span></p></td><td class="c17"><p class="c4"><span>Leu-6(A)</span></p></td><td class="c17"><p class="c4"><span>Leu-6(A)</span></p></td><td class="c23"><p class="c4"><span>13(A)</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>DNA(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c23"><p class="c4"><span>16</span></p></td><td class="c14"><p class="c4"><span>13.5</span></p></td><td class="c43"><p class="c4"><span>6.5</span></p></td><td class="c17"><p class="c4"><span>13..5</span></p></td><td class="c17"><p class="c4"><span>13.5</span></p></td><td class="c23"><p class="c4"><span>12</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>Buffer (</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c23"><p class="c4"><span>2</span></p></td><td class="c14"><p class="c4"><span>2</span></p></td><td class="c43"><p class="c4"><span>2</span></p></td><td class="c17"><p class="c4"><span>2</span></p></td><td class="c17"><p class="c4"><span>2</span></p></td><td class="c23"><p class="c4"><span>2</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>ddH2O(</span><span class="c3">&micro;</span><span>l)</span></p></td><td class="c23"><p class="c4"><span>0</span></p></td><td class="c14"><p class="c4"><span>2.5</span></p></td><td class="c43"><p class="c4"><span>9.5</span></p></td><td class="c17"><p class="c4"><span>2.5</span></p></td><td class="c17"><p class="c4"><span>2.5</span></p></td><td class="c23"><p class="c4"><span>4</span></p></td></tr><tr class="c12"><td class="c17"><p class="c4"><span>enzyme</span></p></td><td class="c23"><p class="c4"><span>EcoRI1</span></p></td><td class="c14"><p class="c4"><span class="c7">Xba</span><span class="c3">&#8544;1</span></p></td><td class="c43"><p class="c4"><span>EcoRI 1</span></p></td><td class="c17"><p class="c4"><span class="c7">Xba</span><span class="c3">&#8544;1</span></p></td><td class="c17"><p class="c4"><span>EcoRI 1</span></p></td><td class="c23"><p class="c4"><span class="c7">Xba</span><span class="c3">&#8544;1</span></p></td></tr><tr class="c12"><td class="c17"><p class="c2"><span class="c3"></span></p></td><td class="c23"><p class="c4"><span class="c7">Spe</span><span class="c3">&#8544;1</span></p></td><td class="c14"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;1</span></p></td><td class="c43"><p class="c4"><span class="c7">Spe</span><span class="c3">&#8544;1</span></p></td><td class="c17"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;1</span></p></td><td class="c17"><p class="c4"><span class="c7">Spe</span><span class="c3">&#8544;1</span></p></td><td class="c23"><p class="c4"><span class="c7">Pst</span><span class="c3">&#8544;1</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.z9hgdmqritzr"></a><span>Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><a href="#" name="id.6d6a1369bdb4"></a><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span class="c27 c3">n</span><span class="c27 c7">g/</span><span class="c27 c3">&micro;</span><span class="c27 c7">l</span></p></td><td class="c9"><p class="c4"><span class="c7">A260</span><span class="c3">&#8725;</span><span class="c7">A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>C0076</span></p></td><td class="c9"><p class="c4"><span>21</span></p></td><td class="c9"><p class="c4"><span>2.154</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Clue-6</span></p></td><td class="c9"><p class="c4"><span>72.5</span></p></td><td class="c9"><p class="c4"><span>1.933</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>Ce0020</span></p></td><td class="c9"><p class="c4"><span>22</span></p></td><td class="c9"><p class="c4"><span>2.064</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>6</span></p></td><td class="c9"><p class="c4"><span>82</span></p></td><td class="c9"><p class="c4"><span>2.130</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>13</span></p></td><td class="c9"><p class="c4"><span>82</span></p></td><td class="c9"><p class="c4"><span>1.864</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>45</span></p></td><td class="c9"><p class="c4"><span>1.738</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.30jo6d81y85k"></a><span>9.24</span></h1><h3 class="c4"><a name="h.loc88q13v29m"></a><span>First ligation</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c4 c22"><span>number</span></p></td><td class="c10"><p class="c4 c22"><span>1</span></p></td><td class="c10"><p><span></span></p></td><td class="c10"><p class="c4 c22"><span>2</span></p></td><td class="c16"><p><span></span></p></td><td class="c10"><p class="c4 c22"><span>3</span></p></td><td class="c16"><p><span></span></p></td></tr><tr class="c12"><td class="c5"><p class="c4 c22"><span>part</span></p></td><td class="c10"><p class="c4"><span>18-3-11 </span></p></td><td class="c10"><p class="c4"><span>2.5</span></p></td><td class="c10"><p class="c4"><span>13</span></p></td><td class="c16"><p class="c4"><span>2</span></p></td><td class="c10"><p class="c4"><span>C0076</span></p></td><td class="c16"><p class="c4"><span>5</span></p></td></tr><tr class="c12"><td class="c5"><p class="c2"><span></span></p></td><td class="c10"><p class="c4"><span>Leu-6</span></p></td><td class="c10"><p class="c4"><span>2</span></p></td><td class="c10"><p class="c4"><span>Leu-6</span></p></td><td class="c16"><p class="c4"><span>2</span></p></td><td class="c10"><p class="c4"><span>6</span></p></td><td class="c16"><p class="c4"><span>3</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4 c22"><span>cariier</span></p></td><td class="c10"><p class="c4 c22"><span>AC 3.5</span></p></td><td class="c10"><p><span></span></p></td><td class="c10"><p class="c4 c22"><span>AC 3.5</span></p></td><td class="c16"><p><span></span></p></td><td class="c10"><p class="c2"><span></span></p></td><td class="c16"><p><span></span></p></td></tr><tr class="c12"><td class="c5"><p class="c4 c22"><span>buffer</span></p></td><td class="c10"><p class="c4 c22"><span>2</span></p></td><td class="c10"><p><span></span></p></td><td class="c10"><p class="c4 c22"><span>2</span></p></td><td class="c16"><p><span></span></p></td><td class="c10"><p class="c4 c22"><span>2</span></p></td><td class="c16"><p><span></span></p></td></tr><tr class="c12"><td class="c5"><p class="c4 c22"><span>T4</span></p></td><td class="c10"><p class="c4 c22"><span>1</span></p></td><td class="c10"><p><span></span></p></td><td class="c10"><p class="c4 c22"><span>1</span></p></td><td class="c16"><p><span></span></p></td><td class="c10"><p class="c4 c22"><span>1</span></p></td><td class="c16"><p><span></span></p></td></tr><tr class="c12"><td class="c5"><p class="c4 c22"><span>ddH2O</span></p></td><td class="c10"><p class="c4 c22"><span>9</span></p></td><td class="c10"><p><span></span></p></td><td class="c10"><p class="c4 c22"><span>9.5</span></p></td><td class="c16"><p><span></span></p></td><td class="c10"><p class="c4 c22"><span>9</span></p></td><td class="c16"><p><span></span></p></td></tr></tbody></table><h1 class="c49 c4"><a name="h.mnap6p5gspdc"></a></h1><h1 class="c4"><a name="h.mnap6p5gspdc"></a><span>9.25</span></h1><h3 class="c4"><a name="h.r2p4yasvsr2t"></a><span>Second ligation</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4 c22"><span>number</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p><span></span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c6"><p><span></span></p></td></tr><tr class="c12"><td class="c9"><p class="c4 c22"><span>part</span></p></td><td class="c0"><p class="c4"><span>E0020</span></p></td><td class="c6"><p class="c4"><span>9</span></p></td><td class="c0"><p class="c4"><span>5-4</span></p></td><td class="c6"><p class="c4"><span>4</span></p></td></tr><tr class="c12"><td class="c9"><p class="c2"><span></span></p></td><td class="c0"><p class="c4"><span>6</span></p></td><td class="c6"><p class="c4"><span>9</span></p></td><td class="c0"><p class="c4"><span>Leu-6</span></p></td><td class="c6"><p class="c4"><span>2.5</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4 c22"><span>cariier</span></p></td><td class="c0"><p class="c2"><span></span></p></td><td class="c6"><p><span></span></p></td><td class="c0"><p class="c4"><span>AK 1.5</span></p></td><td class="c6"><p><span></span></p></td></tr><tr class="c12"><td class="c9"><p class="c4 c22"><span>buffer</span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c6"><p><span></span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c6"><p><span></span></p></td></tr><tr class="c12"><td class="c9"><p class="c4 c22"><span>T4</span></p></td><td class="c0"><p class="c4"><span>1.5</span></p></td><td class="c6"><p><span></span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c6"><p><span></span></p></td></tr><tr class="c12"><td class="c9"><p class="c4 c22"><span>ddH2O</span></p></td><td class="c0"><p class="c4"><span>5.5</span></p></td><td class="c6"><p><span></span></p></td><td class="c0"><p class="c4"><span>9</span></p></td><td class="c6"><p><span></span></p></td></tr></tbody></table><h3 class="c4"><a name="h.wm6ppyi2kw84"></a><span>Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c0"><p class="c4"><span>number</span></p></td><td class="c0"><p><span></span></p></td><td class="c9"><p class="c4"><span class="c27 c3">n</span><span class="c27 c7">g/</span><span class="c27 c3">&micro;</span><span class="c27 c7">l</span></p></td><td class="c9"><p class="c4"><span class="c7">A260</span><span class="c3">&#8725;</span><span class="c7">A280</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>3</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>29</span></p></td><td class="c9"><p class="c4"><span>2.041</span></p></td></tr><tr class="c12"><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>38</span></p></td><td class="c9"><p class="c4"><span>2.171</span></p></td></tr><tr class="c12"><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>35</span></p></td><td class="c9"><p class="c4"><span>2.265</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>E0020</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>69</span></p></td><td class="c9"><p class="c4"><span>1.816</span></p></td></tr><tr class="c12"><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>31</span></p></td><td class="c9"><p class="c4"><span>1.956</span></p></td></tr><tr class="c12"><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>45</span></p></td><td class="c9"><p class="c4"><span>1.957</span></p></td></tr><tr class="c12"><td class="c0"><p class="c4"><span>5-4</span></p></td><td class="c0"><p class="c4"><span>1</span></p></td><td class="c9"><p class="c4"><span>110.5</span></p></td><td class="c9"><p class="c4"><span>1.939</span></p></td></tr><tr class="c12"><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c4"><span>2</span></p></td><td class="c9"><p class="c4"><span>152.0</span></p></td><td class="c9"><p class="c4"><span>1.900</span></p></td></tr><tr class="c12"><td class="c0"><p class="c2"><span></span></p></td><td class="c0"><p class="c4"><span>3</span></p></td><td class="c9"><p class="c4"><span>76.5</span></p></td><td class="c9"><p class="c4"><span>1.987</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.dlir95pkge4n"></a><span>9.26</span></h3><h3 class="c4"><a name="h.aift0ymnjhti"></a><span>Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span class="c27 c3">n</span><span class="c27 c7">g/</span><span class="c27 c3">&micro;</span><span class="c27 c7">l</span></p></td><td class="c9"><p class="c4"><span class="c7">A260</span><span class="c3">&#8725;</span><span class="c7">A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>14-19</span></p></td><td class="c9"><p class="c4"><span>112.5</span></p></td><td class="c9"><p class="c4"><span>1.875</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>RFP-1</span></p></td><td class="c9"><p class="c4"><span>145.0</span></p></td><td class="c9"><p class="c4"><span>1.859</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>RFP-2</span></p></td><td class="c9"><p class="c4"><span>138.5</span></p></td><td class="c9"><p class="c4"><span>1.884</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>RFP-3</span></p></td><td class="c9"><p class="c4"><span>162.0</span></p></td><td class="c9"><p class="c4"><span>1.873</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h3 class="c4"><a name="h.fe703gdi9fmv"></a><span>9.27</span></h3><h3 class="c4"><a name="h.d0esydvlvvow"></a><span>Measure density</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c9"><p class="c4"><span>number</span></p></td><td class="c9"><p class="c4"><span class="c3 c27">n</span><span class="c27 c7">g/</span><span class="c27 c3">&micro;</span><span class="c27 c7">l</span></p></td><td class="c9"><p class="c4"><span class="c7">A260</span><span class="c3">&#8725;</span><span class="c7">A280</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>C1</span></p></td><td class="c9"><p class="c4"><span>18.0</span></p></td><td class="c9"><p class="c4"><span>2.022</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>C2</span></p></td><td class="c9"><p class="c4"><span>26.0</span></p></td><td class="c9"><p class="c4"><span>2.097</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>C3</span></p></td><td class="c9"><p class="c4"><span>25.0</span></p></td><td class="c9"><p class="c4"><span>2.041</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>C4</span></p></td><td class="c9"><p class="c4"><span>23.0</span></p></td><td class="c9"><p class="c4"><span>1.862</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>rhiR</span></p></td><td class="c9"><p class="c4"><span>18.5</span></p></td><td class="c9"><p class="c4"><span>1.900</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>leuB</span></p></td><td class="c9"><p class="c4"><span>12.1</span></p></td><td class="c9"><p class="c4"><span>2.000</span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>14-119-13</span></p></td><td class="c9"><p class="c4"><span>9.6</span></p></td><td class="c9"><p class="c2"><span></span></p></td></tr><tr class="c12"><td class="c9"><p class="c4"><span>RFP</span></p></td><td class="c9"><p class="c4"><span>12.4</span></p></td><td class="c9"><p class="c4"><span>2.041</span></p></td></tr></tbody></table><p class="c2"><span></span></p><h1 class="c4"><a name="h.kd1630mtylul"></a><span>9.30</span></h1><h3 class="c4"><a name="h.31j8dlpozyv4"></a><span>1. measure the density of 3 and digest</span></h3><h3 class="c4"><a name="h.31j8dlpozyv4"></a><span>Density &nbsp;(1) 78 ng/ul &nbsp; (2)62ng/ul</span></h3><h3 class="c4"><a name="h.s92naqwi7onn"></a><span>Digestion</span></h3><p class="c2"><span></span></p><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><h3 class="c4"><a name="h.31j8dlpozyv4"></a><span>DNA</span></h3></td><td class="c5"><p class="c4"><span>Buffer</span></p></td><td class="c13"><p class="c4"><span>XbaI</span></p></td><td class="c13"><p class="c4"><span>PstI</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>16</span></p></td><td class="c5"><p class="c4"><span>2</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td><td class="c13"><p class="c4"><span>1</span></p></td></tr></tbody></table><h3 class="c4"><a name="h.lfmh1js0pg9v"></a><span>2.measure density of 14&amp;19</span></h3><h3 class="c4"><a name="h.lfmh1js0pg9v"></a><span>(1)112ng/ul &nbsp; (2)84ng/ul</span></h3><h3 class="c4"><a name="h.lfmh1js0pg9v"></a><span>10.1</span></h3><h3 class="c4"><a name="h.lfmh1js0pg9v"></a><span>1.measure density</span></h3><table cellpadding="0" cellspacing="0" class="c28"><tbody><tr class="c12"><td class="c5"><p class="c4"><span>3.2</span></p></td><td class="c5"><p class="c4"><span>3.3</span></p></td><td class="c13"><p class="c4"><span>C0076&amp;6</span></p></td><td class="c13"><p class="c4"><span>C0076&amp;6</span></p></td></tr><tr class="c12"><td class="c5"><p class="c4"><span>273.5</span></p></td><td class="c5"><p class="c4"><span>275</span></p></td><td class="c13"><p class="c4"><span>164</span></p></td><td class="c13"><p class="c4"><span>169</span></p></td></tr></tbody></table><p class="c2"><span></span></p><p class="c2"><span></span></p>
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Revision as of 15:34, 5 October 2011

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7.21

I  Prepare medium

1.soc medium

2.LB medium

3.LBliquid medium

II  transform 2011 parts –OUC Order 1 to 3

III  pick up a tested single colony

7.22

1.Extract plasmid of the practicing part

2. Measure density using distilled water as comparison

A260/A280

A260/A230

density(ng/ul)

1

1.892

2.115

0.035

2

1.841

1.227

0.0405

3

2.299

4.054

0.030

3. Digestion for 3 hours

1

2

3

4(Single enzyme cut)

DNA(ul)

15

15

15

15

Buffer2(ul)

5

5

5

5

Bsa(ul)

0.5

0.5

0.5

-----

Double-distilled water(ul)

27.5

27.5

27.5

29

EcoR I(ul)

1

1

1

1

Pst I(ul)

1

1

1

-------

Time(h)

3

3

3

3

4.check by Electrophoresis

1、2、3:double enzyme,4、5、6:Single enzyme ,7:plasmid

5.transform 7new parts(number:4-10)

6.culture bacteria on LB medium

7.culture 3 new parts in Shaker

7.24

1. Presrvation species of parts No. ,4,5,6,7,8,9,10

2. Extract plasmid of 4,5,6,7,8,9,10

3.check through double enzyme Digestion

7.25

1. Measure density

5

0.020µg/µl

4

0.012µg/µl

6

0.020µg/µl

2

0.015µg/µl

2. Digestion

5

4

6

2

DNA

25

30

25

25

Buffer2

5

5

5

5

BSA

0.5

0.5

0.5

0.5

H2O

17.5

12.5

17.5

17.5

enzyme1

EcoRⅠ1µl

EcoRⅠ1µl

SpeⅠ1µl

XbaⅠ1µl

Enzyme2

SpeⅠ1µl

XbaⅠ1µl

PstⅠ1µl

PstⅠ1µl

3.check by Electrophoresis

4.gel extraction

5.measure density

OUC-order

5

4

6

2

Density  ug/µl

0.016

0.021

0.027

0.028

6. Ligate overnight

1

2

Parts

2µl 5+3µl 4

3µl 2+3µl 6

Buffer

2µl

2µl

enzyme

1µl

1µl

DdH2O

2µl

1µl

7.28

Repeat the ligation expriments of 7.25

7.29

Extract plasmids of parts No. 11,12,13,14

7.30

Medium has antibiotic resistance problem, re-transform 11\12\13\14

7.31

1.cultrue bacteria of 11,12,13,14 in LB liquid medium

2. Extract plasmid of 15,16,17,18,19,20,21,22

3. Optimization the Steps of Extracting plasmid

4.Chloramphericol preparing

5. NaAC’s DNA concentration

8.1

1. Extract plasmid of 11-14 and Measure density

ng/µl

A260∕A280

11

12.7

1.917

12

10.3

1.981

13

35.5

1.919

14

15.7

1.975

2. Extract plasmid of No.5,4,6,2,15-20 parts

3.culture the colony of GFP on LB medium

8.2

Measure density of 15-22,2,4,5,6

Parts

µg/µl

A260∕A280

2

0.038

1.875

4

0.019

1.298

5

0.022

1.538

6

0.020

1.702

15

0.012

1.237

16

0.024

1.649

17

0.018

1.731

18

0.015

1.470

21

0.017

1.538

8.3

1. Concentrate plasmid using alcohol and NaAC

2. Measure density after concentrating

ng/µl

A260∕A280

2

103

1.807

4

33

1.811

5

56

1.836

6

41

1.745

8.4

Digestion

Double enzymes

Single enzyme

Parts

5

2

4

6

DNA

25

13

27

25

Buffer

5

5

5

5

BSA

0.5

0.5

0.5

0.5

ddH2O

16.5

28.5

16.5

18.5

enzyme

EcoRⅠ 1.5

XbaⅠ1.5

XbaⅠ1

SpeⅠ1

SpeⅠ1.5

PstⅠ1.5

Parts

4

6

DNA

28.5

30

Buffer

EcoRⅠbuffer 5

Buffer3 5

BSA

0.5

ddH2O

15.5

13.5

enzyme

EcoRⅠ1

PstⅠ1

8.5

1. Begin device 2

2. Design experimental procedures:

3. First construction with parts of device 2

8.6

2+6 successfully ligate

Measure density

Parts

µg/µl

A260∕A280

3

0.085

1.910

16

0.119

1.867

18

0.019

2.033

Measure density after concentrating

Parts

µg/µl

A260∕A280

18

0.0605

1.921

11

0.0335

2.107

Digestion

Double enzymes

Single enzyme

Parts

18

16

3

11

DNA

20

10

15

29

Buffer

5

5

5

5

BSA

0.5

0.5

0.5

0.5

ddH2O

21.5

31.5

28

14.5

enzyme

EcoRⅠ 1.5

XbaⅠ1.5

XbaⅠ1

SpeⅠ1

SpeⅠ1.5

PstⅠ1.5

After 8hours

Parts

3

11

DNA

20

20

Buffer

EcoRⅠbuffer 5

Buffer3 5

BSA

0.5

ddH2O

23.5

23

enzyme

EcoRⅠ1

PstⅠ1

8.7

1. Checking 4+5, 2+6 ‘s ligation by Gel electrophoresis

 Digestion

μL

2+6

4+5

DNA

2(53ng)

3(28ng)

Buffer2

1

1

BSA

0.1

0.1

ddH2O

6.7

5.9

EcoRⅠ

0.1

0.1

PstⅠ

0.1

0.1

check by small gel

8.8

1.transform the parts of 3-18,11-16,14-19

2. Active bacteria of 4.5.18.11 and culture in LB liquid medium

3. Presrvation species parts of 4-5

8.9

1.check 2+6,4+5 by small gel

2. Measure density

number

ng/µl

A260∕A280

2+6

1

58

1.902

2

78.5

1.826

3

62.5

1.953

4

60.5

2.017

4+5

1

31

2.081

2

33

2.089

3

20.5

2.291

3. Digestion

parts

2+6

5+4

number

1

2

3

4

1

2

3

DNA

2

2

2

2

3

3

5

Buffer3

1

1

1

1

1

1

1

BSA

0.1

0.1

0.1

0.1

0.1

0.1

0.1

ddH2O

6.5

6.5

6.5

6.5

5.5

5.5

5.5

EcoRⅠ

0.2

0.2

0.2

0.2

0.2

0.2

0.2

PstⅠ

0.2

0.2

0.2

0.2

0.2

0.2

0.2

4. Extract plasmid of  4,5,18,11

Measure density

number

ng/µl

A260∕A280

5

73

2.000

4

71.5

2.072

18

32.5

2.305

3

85

1.910

11

95.5

1.950

16

119

1.867

Digestion 50µl

5

4

18

3

11

16

DNA

13

13

30

11

10

8

Buffer3

5

5

5

5

5

5

BSA

0.5

0.5

0.5

0.5

0.5

0.5

ddH2O

29.5

29.5

19.5

31.5

30.5

34.5

enzyme

EcoRⅠ 1

XbaⅠ1

EcoRⅠ 1

XbaⅠ1

EcoRⅠ 1

XbaⅠ1

SpeⅠ1

PstⅠ1

SpeⅠ1

PstⅠ1

SpeⅠ1

PstⅠ1

8.11

1. Checking ligation correctness of 14&19 using small gel----fails

2.try to ligate using 3A

8.12

1. Extract plasmid of  5+4 and Measure density

number

ng/µl

A260∕A280

1

55

1.964

2

39

2.324

3

41

2.412

4

81

2.051

5

42

2.154

6

58

1.966

2.check by single enzyme digestion

number

1

2

3

4

5

6

DNA

3

4

4

2

4

3

Buffer EcoR I

1

1

1

1

1

1

ddH2O

5.5

4.5

4.5

6.5

4.5

5.5

EcoRⅠ

0.5

0.5

0.5

0.5

0.5

0.5

Check using small gel

3.cultrue bacteria of part 13

4. ligate  2+6+3,11+16,18+13

8.13

1.Extract plasmid

2.pick colony of 2+6+3,18+3,16+3 and culture in shaker

8.14

Checking whether colony of different size contain different plasmids, or whether could be used to pick colony raising transformation possibility. But it made no difference

8.15

1.measure density

number

µg/µl

A260∕A280

2+6+3-1

0.032

3.439

2+6+3-2

0.064

2.370

11+16

0.056

2.306

11+16

0.102

2.010

18+3

0.037

2.434

18+3

0.033

2.332

2. Digestion

Single enzyme 10µl

number

2+6+3-1

2+6+3-2

3+18-1

3+18-2

11+16

11+16

DNA

4

2

4

6

2.5

1.5

Buffer EcoR I

1

1

1

1

1

1

ddH2O

4.7

6.7

4.7

2.7

6.2

7.2

EcoRⅠ

0.3

0.3

0.3

0.3

0.3

0.3

Double enzyme 10µl

number

18+3-1

18+3-2

18

2+6+3-1

2+6+3-2

2+6

11+16

11+16

DNA

4

6

6

4

2

3

2.5

1.5

Buffer3

1

1

1

1

1

1

1

1

BSA

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

ddH2O

4.3

2.3

2.4

4.3

6.3

5.3

5.8

6.8

EcoRⅠ

0.3

0.3

0.3

0.3

0.3

0.3

0.3

0.3

PstⅠ

0.3

0.3

0.3

0.3

0.3

0.3

0.3

0.3

8.16

Check yesterdays’ digestion by gel electrophoresis

Ladder

18-3

18-3

18

6-2-3

6-2-3

6-2

100bp plus

single

double

single

double

double

double

double

double

8.17

1. Presrvation species of pSB1AC3_BBa13017

2. Extract plasmid of 18,pSB1AC3_BBa13017,measure density

number

µg/µl

A260∕A280

pSB1AC3_BBa13017-1

0.199

2.036

pSB1AC3_BBa13017-2

0.168

2.036

pSB1AC3_BBa13017-3

0.113

2.083

pSB1AC3_BBa13017-4

0.103

2.102

pSB1AC3_BBa13017-5

0.174

2.129

pSB1AC3_BBa13017-6

0.147

2.115

pSB1AC3_BBa13017-7

0.064

2.345

pSB1AC3_BBa13017-8

0.106

2.186

pSB1AC3_BBa13017-9

0.163

2.138

pSB1AC3_BBa13017-10

0.170

2.092

18-1

0.110

2.095

18-2

0.124

2.102

18-3

0.140

2.121

3. Prepare medium  of  TY(culturing rhizobium),CM(supplemented medium)

4. Digest pSB1AC3_BBa13017 as Carrier

DNA

5

Buffer2

5

BSA

0.5

ddH2O

37.5

EcoRⅠ

1

PstⅠ

1

Extract 3055bp strip

8.18

1.Ligate 5,4

2.transform  pSB1C3

8.19

1.culture rhizobium on TY medium

2.prepare CM,M9 medium

3.LeuB PCR

4. Presrvation species of Bean strain

5. Extract plasmid and Digest

number

18

3

2+6

16

11

14+19

13

DNA

12

10

18

9

7

7

6

Buffer2

5

5

5

5

5

5

5

BSA

0.5

0.5

0.5

0.5

0.5

0.5

0.5

ddH2O

30.1

31.5

24.1

33.1

35.1

35.1

36.1

enzyme

EcoRI1.2

XbaⅠ1.2

EcoRI1.2

XbaⅠ1.2

EcoRI1.2

EcoRI1.2

XbaⅠ1.2

SpeⅠ1.2

PstⅠ1.2

SpeⅠ1.2

PstⅠ1.2

SpeⅠ1.2

SpeⅠ1.2

PstⅠ1.2

8.20

1. Presrvation species of 5+4 and extract plasmid,measure density

number

ng/µl

A260∕A280

1

99.5

2.098

2

140.5

2.162

3

99

2.152

4

123.5

2.093

5

137.5

2.083

2. Digestion

number

1

2

3

4

5

DNA

1.3

1

1.3

1

1

Buffer3

1

1

1

1

1

BSA

0.1

0.1

0.1

0.1

0.1

ddH2O

7.1

7.4

7.1

7.4

7.4

EcoRⅠ

0.25

0.25

0.25

0.25

0.25

PstⅠ

0.25

0.25

0.25

0.25

0.25

8.22

1、Presrvation species of 5+4 and extract plasmid,measure density, digest

2、pick single colonies of 19-14&13;2-6&3 , 11&16 and culture in shaker

3、PCR Leu(50ul),recover and check

1Taq Buffer(with MgSO4) 5μL

MgCl2  4μL

Primer-F 1.5μL

Primer-R 1.5μL

H2O 33.5μL

dNTP 2μL

DNA 2μL

Taq 0.25μL

Pfu 0.25μL                                      

Recommended By Advicer Tan

Result: success

8.23

1、Presrvation species of 2 6&3,11&16,14 19&13 and extract plasmid, measure density,digest

density

number

2 6&3

11&16

1419&13

density

(ng/ul)

30

49

45

132

62

42

64

Digestion

Single enzyme

2 6&3

11&16

1419&13

DNA

3.3

2

2.2

1

1.5

2.3

1.5

Buffer4

1

1

1

1

1

1

1

DdH2O

5.4

6.6

6.4

7.6

7.1

6.3

7.1

XbaI

0.3

0.3

0.3

0.3

0.3

0.3

0.3

BSA

0.1

0.1

0.1

0.1

0.1

0.1

0.1

Double enzymes

2 6&3

11&16

1419&13

DNA

3.3

2

2.2

1

1.5

2.3

1.5

Buffer4

1

1

1

1

1

1

1

BSA

0.1

0.1

0.1

0.1

0.1

0.1

0.1

H2O

5.1

6.4

6.2

7.4

6.9

6.1

6.9

EcoRI-HF

0.25

0.25

0.25

0.25

0.25

0.25

0.25

SpeI

0.25

0.25

0.25

0.25

0.25

0.25

0.25

result:success

2、Preservation species of CFN42 WT and BL21

3、check 18&3 be small gel ——succeed

4、Prepare medium:LB liquid medium  300ml,

LB medium 150ml

                     TY medium 150ml

5、Prepare SMM medium,+ pro+ Vb1

8.24

1、Prepare SMM medium  :MM+pro+Vb1  MM+pro+Vb1+leu

2、check by small gel

8.25

1、ligate 11-16&18-13 and 5&4 using 3A

Measure density:

11-16

18-13

5

4

density(ng/ul)

46.5

136.5

85.5

71

Double enzymes

5

4

18-3

11-16

DNA

12

10

10

25

Buffer4

5

5

5

5

BSA

0.5

0.5

0.5

0.5

H2O

29.5

32.5

31.5

16.5

enzyme1

EcoRI-HF 1.5

XbaI  1

EcoRI-HF  1.5

XbaI  1.5

enzyme 2

SpeI 1.5

PstI  1

SpeI  1.5

PstI  1.5

Check by gel

Ligation of 5&4

5

4

AC

T4

Buffer

H2O

6

4

4

1

2

3

Ligation of  11-16&18-13:(ul)

K381001

18-3

11-16

Buffer

T4

5

6

6

2

1

2、inoculate HB101 and BL21 into the solid medium (MM+VB1+Pro and MM+VB1+Pro+Leu),culture the Rhizobium etli CFN42 and Sinorhizobium meliloti 1021 on TY solid medium at 30℃

8.27

1、transform 18-3&11

2、pick 3 colonies of 5&4 and culture in l LB liquid medium

3、culture LeuB and K115001 in LB liquid medium

4、recover 22,21,3 and ligate 21&3,22&3

Ligation of 21&3:(ul)

22

3

T4

Buffer

ddH2O

7

4

1

2

6

Ligation of  22&3:(ul)

21

3

T4

Buffer

ddH2O

8.5

4

1

2

4.5

8.28

1、Prepare medium:LB liquid medium  500ml

2、transform 5&4 and 18-3&11-16 again,and transform leu 21&3 and 22&3

3、 culture deficient cells

4、culture 18-3&11 in LB liquid medium

8.29

1、PCR :5&4 and leu

PCR(ul)

ddH2O

dNTP

Buffer

Primer-F

Primer-R

dna

Taq

39.5

2

5

1

1

1

0.5

2、Presrvation species of 18-3&11 and extract plasmid,measure density, digest

density(1)19.5(2)130.5

digestion

single enzyme

(1)

(2)

DNA

6

1

Buffer

1

1

BSA

0.1

0.1

ddH2O

2.65

7.65

XbaI

0.35

0.35

Double enzymes:

(1)

(2)

DNA

6

1

Buffer

1

1

BSA

0.1

0.1

ddH2O

2.4

7.4

EcoRI

0.25

0.25

PstI

0.25

0.25

Check by small gel

3、pick single colony of 5&4,leu,21&3,22&3,18-3&11-16 and culture in LB liquid medium。

4、prepare TY medium 100ml

8.30

1、Presrvation species of 5&4,leuB89,leuB13 and extract plasmid,measure density, digest

density:

leuB13(1)

5&4(1)

5&4(2)

5&4(3)

leuB89(1)

leuB89(2)

leuB89(3)

24.5

188.0

62.0

132.0

6.8

3.1

10.8

Digestion of  5&4

(1)

(2)

(3)

DNA

1

2

1

Buffer3

1

1

1

ddH2O

7.5

6.5

7.5

BSA

0.1

0.1

0.1

EcoRI

0.2

0.2

0.2

PstI

0.2

0.2

0.2

Check by small gel

Result:5&4 and leuB successfully ligate

8.31

1、did enzyme digestion to 2 part (with psB1AK2 carrier) and K115001 part(with psB1AT3 carrier) and run electrophoresis together with psB1C3. We recycled the carrier part by cutting the gel.

density:

2 part   37.5

K115001 part   120

digestion(ul)

K115001

2

DNA

9

9

10*H Buffer

2

2

EcoRI

1

1

PstI

1

1

ddH2O

7

7

9.1

1、prepare 0147TY medium

Tryptone  5.0g  Yeast extract 3.0g  CaCl.6H2O  1.3g  Agar  15.0g Distilled water 1.0L

2、ligate 18-3&11-16 and 18-3&11

density:

18-3(1)

18-3(2)

11-16(1)

11-16(2)

K carrier(1)

K carrier(2)

AC(1)

AC(2)

140

98

278.5

203.0

17.5

17.5

7.9

13.1

Digestion:

18-3

11

11-16

DNA

7

10

4

Buffer

2

2

2

enzyme1

E  1

X   1

X  1

enzyme 2

S  1

P    1

P   1

DdH2O

9

6

12

Ligation:

18-3:3ul  16:1ul   2:6ul  T4:1ul  Buffer:2ul   H2O:7ul

18-3:3ul  11-16:4ul  2:6ul   T4:1ul   Buffer:2ul  ddH2O:4ul

9.2

1、Presrvation species of leuB and extract plasmid,measure density, digest

density:

1

2

3

4

292.5

21.5

223.5

32.0

Digestion:

leuB

K(pSB1C3)

EcoRI

1

1

PstI

1

1

Buffer

2

2

DNA

14

8.5

ddH2O

2

7.5

Check by small gel:

recover:

2、pick single colony of 18-3&11-16 and 18-3&11 and culture in shaker

3、transform BBa_I751250,as LuxI_AHL producer。Transform BBa_J04450(placI+mRFP)

9.3

1、ligate leuB, K(pSB1C3) carrier and transform

density:

Before Concentration

After  Concentration

LeuB

15

36

pSB1C3

8

25

ligation

LeuB

Psb1c3

T4

Buffer

DdH2O

6

4

1

2

7

2、extract plasmid of 8-3&11-16 and 18-3&11, digest,and check by small gel

Density:

18-3&11-161

18-3&11-162

18-3&11 1

18-3&11 2

5-4(AC)1

5-4(AC) 2

2-6-3

265

242

192

217.5

193

72

63.5

digestion:

18-3&11-16(1)

18-3&11-16(2)

18-3&11(1)

18-3&11(2)

DNA

3.5

4

5

4.5

Buffer

2

2

2

2

EcoRI

1

1

1

1

PstI

1

1

1

1

ddH2O

12.5

12

11

11.5

Check by gel:

2、ligate 5-4&2-6-3

ligation

K

5-4

2-6-3

T4连接酶

Buffer

DdH2O

6

4

2

1

2

5

9.4

1、transform 5-4&2-6-3,extract plasmid of  2-6-3与2

2、transform Pea Rhizobium

9.5

1、Presrvation species of leuB and extract plasmid,measure density, digest, PCR ,check by gel

PCR

1

2

3

ddH2O

37.5

37

36.5

36.5

36.5

dNTPmix

4

4

4

4

4

Buffer

5

5

5

5

5

VF2

1.5

1.5

1.5

1.5

1.5

VR

1.5

1.5

1.5

1.5

1.5

tamplate

0

0.5

1

1

1

Taq

0.5

0.5

0.5

0.5

0.5

Check by gel

digestion

LeuB  1

LeuB  3

DNA

3

3

BufferM

0.5

0.5

ddH2O

1.1

1.1

SpeI

0.2

0.2

XbaI

0.2

0.2

Check by gel

9.6

1、Presrvation species of 54263 and extract plasmid, digest, PCR ,check by gel

digestion

1

2

3

DNA

1.4

1.4

1.7

Buffer

0.5

0.5

0.5

DdH2O

2.5

2.5

2.2

EcoRI

0.3

0.3

0.3

PstI

0.3

0.3

0.3

9.7

1、measure OD of Deficient cell

MM(leuB-)

1  leuB+

2   leuB+

3   leuB+

4  leuB+

OD(A340)

1.611-1

1.698-1

1.818-1

1.548-1

1.881-1

2、digest  leuB、③、K115001

digestion:

8

6

LeuB

3

T1

T2

T3

DNA

9

9

10

10

10

13

12

Buffer

2

2

2

2

2

2

2

DdH2O

7

6.6

5.6

5.6

5.6

2.6

3.6

EcoRI

1

1.2

1.2

1.2

1.2

1.2

1.2

SpeI

1

1.2

1.2

1.2

1.2

1.2

1.2

density:

LeuB 98.0

T  1:93.5     2:73.5    3:87

3  1:97       2:73     3:82

9.8

1、extract plasmid of 6,and digest with LeuB

Density of 6:

1

2

3

57

68

50

digestion

6

6

LeuB

DNA

6

6

10

Buffer

2

2

2

DdH2O

9.6

9.6

5.6

EcoRI

1.2

1.2

1.2

SpeI

1.2

1.2

1.2

2、ligate leuB&3

ligation:

T4

Buffer

leuB

3

T carrier

DdH2O

1

2

3

0.7

10

3.3

9.9

1、check 6, leuB, 8

Gel:

Ligate 8&6、6&leuB

Ligation of 8&6:

T carrier

8

6

T4

Buffer

DdH2O

10

1

1

1

2

5

Ligation of  6&leuB

T carrier

6

LeuB

T4

Buffer

DdH2O

10

1

3

1

2

3

9.10

1、transform 8&6 and  leuB

2、culture leuB&3 in LB medium

3、device checking:

Device1 has strong Fluorescent and device 2 hasslight one

Picture:

9.11

1、PCR:sinR、pro-RBS-sinR、raiR、raiR-pro

density

sinI

12.8

SinI pro

9.6

Pro-RBS-sinI

15.1

RaiI

14.2

RaiI pro

9.4

Pro-RBS-raiI

11.4

SinR pro

11.5

Pro-RBS-raiR

15.0

2、pick single colony of 6&leuB

9.12

1、digest 21、22 and ligate

digestion

21

22

Buffer

2

2

NDA

13.6

15

DdH2O

2

0.6

EcoRI

1.2

1.2

SpeI

1.2

1.2

ligation

21&3

21

3

Buffer

T4

DdH2O

T

3

1

2

1

3

10

22&3

22

3

Buffer

T4

DdH2O

T

5

1

2

1

1

10

2、extract plasmid of leuB-6 and measure density

density:16.5

9.13

1、transform and culture 21&3,22&3,8&6,recover PSB1C3 carrier

2.Pcr

ddH2O

Pfu with MgSO4 buffer

dNTP

PF

PR

Template

Pfu

38µl

5µl

4µl

1µl

1µl

0.5µl

0.5µl

Results:failed

3.digest sinR, sinI,pro- sinI

Digestion:20µl

sinR

sinI

pro- sinI

DNA

10

11

16

Buffer 10×H

2

2

2

ddH2O

6

5

0

EcoRI

1

1

1

PstI

1

1

1

9.15

Digestion of Leu&3 :20µl

number

1

2

3

DNA(µl)

10

10

9

Buffer 10×H(µl)

2

2

2

ddH2O(µl)

6

6

7

Xba(µl)

1

1

1

Pst(µl)

1

1

1

9.16

Digest 6 and leub

number

6

Leu

DNA(µl)

15

9

Buffer 10×M(µl)

2

2

ddH2O(µl)

2

7

enzymew

Spe(µl) 1

Xba(µl) 1

Pst(µl) 1

9.18

Ligate Leu and 6 using 3A

Leu

6

buffer

T4

10µl

l

l

l

l

Gel picture:

Digest 6

number

DNA(µl)

Buffer 10× (µl)

Pst(µl)

ddH2O(µl)

6

9

2

1

8

9.20

Measure density

number

ng/µl

A260A280

6

179.5

1.851

pSB1C3-1

6.3

pSB1C3-2

8.6

3.510

pSB1C3-3

5.1

Digest Leu

DNA(µl)

Buffer 10×M(µl)

ddH2O(µl)

Pst(µl)

Xba(µl)

6

2

2

2

8

Ligate Leu& 6

Leu

6

buffer

T4

ddH2O

6

4

2

1

7

9.21

Measure density

number

ng/µl

A260A280

raipro-3

29.5

1.666

raipro-2

28.5

1.885

raipro-1

23.0

1.818

C0076

161.0

1.793

E0020e0fp-1

28.5

1.676

E0020e0fp-2

19.5

2.191

Digestion 5µl

number

raipro-1

raipro-2

raipro-3

DNA(µl)

4

4

4

Buffer 10×H (µl)

0.5

0.5

0.5

EcoRI(µl)

0.25

0.25

0.25

PstI(µl)

0.25

0.25

0.25

20µl

number

DNA(µl)

Buffer 10×M(µl)

ddH2O(µl)

Pst(µl)

Xba(µl)

C0076

6.5

2

9.5

1

1

50µl

number

DNA(µl)

Buffer 10×M(µl)

ddH2O(µl)

Pst(µl)

Xba(µl)

E0020

24

5

18

1.5

1.5

9.23

devive

number

5-4(AC)

Leu-6(A)

18-3-11(AK)

Leu-6(A)

Leu-6(A)

13(A)

DNA(µl)

16

13.5

6.5

13..5

13.5

12

Buffer (µl)

2

2

2

2

2

2

ddH2O(µl)

0

2.5

9.5

2.5

2.5

4

enzyme

EcoRI1

XbaⅠ1

EcoRI 1

XbaⅠ1

EcoRI 1

XbaⅠ1

SpeⅠ1

PstⅠ1

SpeⅠ1

PstⅠ1

SpeⅠ1

PstⅠ1

Measure density

number

ng/µl

A260A280

C0076

21

2.154

Clue-6

72.5

1.933

Ce0020

22

2.064

6

82

2.130

13

82

1.864

2

45

1.738

9.24

First ligation

number

1

2

3

part

18-3-11

2.5

13

2

C0076

5

Leu-6

2

Leu-6

2

6

3

cariier

AC 3.5

AC 3.5

buffer

2

2

2

T4

1

1

1

ddH2O

9

9.5

9

9.25

Second ligation

number

1

2

part

E0020

9

5-4

4

6

9

Leu-6

2.5

cariier

AK 1.5

buffer

2

2

T4

1.5

1

ddH2O

5.5

9

Measure density

number

ng/µl

A260A280

3

1

29

2.041

2

38

2.171

3

35

2.265

E0020

1

69

1.816

2

31

1.956

3

45

1.957

5-4

1

110.5

1.939

2

152.0

1.900

3

76.5

1.987

9.26

Measure density

number

ng/µl

A260A280

14-19

112.5

1.875

RFP-1

145.0

1.859

RFP-2

138.5

1.884

RFP-3

162.0

1.873

9.27

Measure density

number

ng/µl

A260A280

C1

18.0

2.022

C2

26.0

2.097

C3

25.0

2.041

C4

23.0

1.862

rhiR

18.5

1.900

leuB

12.1

2.000

14-119-13

9.6

RFP

12.4

2.041

9.30

1. measure the density of 3 and digest

Density  (1) 78 ng/ul   (2)62ng/ul

Digestion

DNA

Buffer

XbaI

PstI

16

2

1

1

2.measure density of 14&19

(1)112ng/ul   (2)84ng/ul

10.1

1.measure density

3.2

3.3

C0076&6

C0076&6

273.5

275

164

169