Team:ZJU-China/August.html
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href="https://2011.igem.org/Team:ZJU-China/August.html">August<br /> | href="https://2011.igem.org/Team:ZJU-China/August.html">August<br /> | ||
</a> <a href="https://2011.igem.org/Team:ZJU-China/September.html">September<br /> | </a> <a href="https://2011.igem.org/Team:ZJU-China/September.html">September<br /> | ||
- | </a></div> | + | </a><a href="https://2011.igem.org/Team:ZJU-China/October.html">October</a></div> |
<h4>Protocol</h4> | <h4>Protocol</h4> | ||
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cellpadding="1"> | cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td width="286"> | + | <td width="286">▪Fusion PCR Vgb+YFP+tetR, NirB+RFP+tetR,</td> |
- | <td width="251"> | + | <td width="251">▪PCR: NirB, Vgb</td> |
</tr> | </tr> | ||
</table> | </table> | ||
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cellpadding="1"> | cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td width="124"> | + | <td width="124">▪Cut: 13K+10I, 22M+10I</td> |
- | <td width="180"> | + | <td width="180">▪Purify: 13K+10I, 22M+10I<br /> |
- | + | ▪Ligation: Pvgb+22M+10I, Pnirb+13K+10I</td> | |
- | <td width="154"> | + | <td width="154">▪PCR backbones<br /> |
- | + | ▪Electrophresis</td> | |
- | <td width="136"> | + | <td width="136">▪Gel excision and purification</td> |
</tr> | </tr> | ||
</table> | </table> | ||
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<table width="611" border="0" cellspacing="1" cellpadding="1"> | <table width="611" border="0" cellspacing="1" cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td width="304"> | + | <td width="304">▪Make Phusion Buffer, isothermel buffer</td> |
- | <td width="300"> | + | <td width="300">▪colony PCR: 20H, 20J, 22B</td> |
</tr> | </tr> | ||
</table> | </table> | ||
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<table width="618" border="0" cellspacing="1" cellpadding="1"> | <table width="618" border="0" cellspacing="1" cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td> | + | <td>▪Cut: 10I+22M, 10I+13; and purification</td> |
- | <td> | + | <td>▪Ligation: Pvgb+22M+10I, Pnirb+13K+10I,<br /> |
- | + | ▪colony PCR: 13K+10I</td> | |
- | <td> | + | <td>▪Culture: 20H, 20J, 22B, 1K, 1I,3C, 5E, 7C</td> |
- | <td> | + | <td>▪Transform: 1G, 3A, 5A, 7A from the distribution plate</td> |
</tr> | </tr> | ||
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<table width="612" border="0" cellspacing="1" cellpadding="1"> | <table width="612" border="0" cellspacing="1" cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td width="188"> | + | <td width="188">▪Check the plates. Contamination, or no |
positive colonies</td> | positive colonies</td> | ||
- | <td width="121"> | + | <td width="121">▪Miniprep: 5 backbones, 22B-1, 22B-3</td> |
- | <td width="197"> | + | <td width="197">▪PCR: nirB from 13K+10I+nirB to firm the |
ligation. One positive result.</td> | ligation. One positive result.</td> | ||
- | <td width="93"> | + | <td width="93">▪Culture the positive colony.</td> |
</tr> | </tr> | ||
</table> | </table> | ||
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<table width="610" border="0" cellspacing="1" cellpadding="1"> | <table width="610" border="0" cellspacing="1" cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td width="244"> | + | <td width="244">▪Cut: 22M,13K with E & S</td> |
- | <td width="164"> | + | <td width="164">▪Culture the red colonies from plate of pSB1C3</td> |
- | <td width="192"> | + | <td width="192">▪PCR: 5 backbones<br /> |
- | + | ▪Cut the PCR products with P+E and run the gel to confirm.</td> | |
</tr> | </tr> | ||
</table> | </table> | ||
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cellpadding="1"> | cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td width="191"> | + | <td width="191">▪The gel results of 5 backbones are right.</td> |
- | <td width="249"> | + | <td width="249">▪Miniprep: pSB1C3, vgb+22M+10I</td> |
- | <td width="157"> | + | <td width="157">▪Culture vgb+22M+1oI in hypoxia<br /> |
- | + | ▪CR pSB1C3</td> | |
</tr> | </tr> | ||
</table> | </table> | ||
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cellpadding="1"> | cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td width="33%"> | + | <td width="33%">▪Run the PCR product</td> |
- | <td width="33%"> | + | <td width="33%">▪PCR pSB1C3 again</td> |
- | <td width="33%"> | + | <td width="33%">▪Run the product, results are good.</td> |
</tr> | </tr> | ||
</table> | </table> | ||
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<table width="100%" border="0" cellspacing="0" cellpadding="1"> | <table width="100%" border="0" cellspacing="0" cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td width="170"> | + | <td width="170">▪Culture: 20H, 20J<br /> |
- | + | ▪Transform 13K from plate 3</td> | |
- | <td width="184"> | + | <td width="184">▪Miniprep: 20H-1, 20J-1</td> |
- | <td> | + | <td>▪Cut: the 20H-1, 20J-1, 22B-1, 22B-3 with E+P</td> |
</tr> | </tr> | ||
</table> | </table> | ||
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<table width="100%" border="0" cellspacing="0" cellpadding="1"> | <table width="100%" border="0" cellspacing="0" cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td> | + | <td>▪Run the digestion results. Bands are confirmed right.</td> |
- | <td> | + | <td>▪Colony PCR vgb+22M+10I</td> |
- | <td> | + | <td>▪Cut the plasmid of vgb+22M+10I</td> |
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<table width="100%" border="0" cellspacing="0" cellpadding="1"> | <table width="100%" border="0" cellspacing="0" cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td> | + | <td>▪Make new stock of competent cells</td> |
- | <td> | + | <td>▪Ligation: 13K+10I<br /> |
- | + | ▪Cut the PCR results and 22M with E+P</td> | |
- | <td> | + | <td>▪Sequence: nirB, vgbL, 12I, 20H, 20J, 22B</td> |
- | <td> | + | <td>▪Transform 13K+10I+pSB1K3<br /> |
- | + | ▪Cut 13K to validate. Results are right.</td> | |
</tr> | </tr> | ||
</table> | </table> | ||
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<table width="620" border="0" cellspacing="0" cellpadding="1"> | <table width="620" border="0" cellspacing="0" cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td> | + | <td>▪Colony PCR: 13K+10I+pSB1K3</td> |
- | <td> | + | <td>▪Culture: vgb+22M+10I in 5ml(shaking), 5ml(water bath), |
10ml(water bath), 15(water bath).</td> | 10ml(water bath), 15(water bath).</td> | ||
- | <td> | + | <td>▪Check the YFP expression of 5ml(shaking) and 15ml(water |
bath). No yellow fluorescent cells,</td> | bath). No yellow fluorescent cells,</td> | ||
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<table width="620" border="0" cellspacing="0" cellpadding="1"> | <table width="620" border="0" cellspacing="0" cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td width="244"> | + | <td width="244">▪Transform: the Gibson assembly results.</td> |
- | <td width="164"> | + | <td width="164">▪Miniprep: 13K+10I<br /> |
- | + | ▪Cut: 13K+10I</td> | |
- | <td> | + | <td>▪Purification: the digestion results.<br /> |
- | + | ▪Ligation: nirB+13K+10I</td> | |
</tr> | </tr> | ||
</table> | </table> | ||
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cellpadding="1"> | cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td width="191"> | + | <td width="191">▪Plate: nirB+13K+10I</td> |
- | <td width="249"> | + | <td width="249">▪Colony PCR: vgb+YFP+tetR +terminater</td> |
- | <td width="157"> | + | <td width="157">▪Gibson PCR</td> |
</tr> | </tr> | ||
</table> | </table> | ||
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cellpadding="1"> | cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td width="140"> | + | <td width="140">▪Colony PCR: vgb+22M+10I, nirB+13K+10I</td> |
- | <td width="181"> | + | <td width="181">▪No bands of 13K; 22M confirmed</td> |
</tr> | </tr> | ||
</table> | </table> | ||
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<table width="609" border="0" cellspacing="1" cellpadding="1"> | <table width="609" border="0" cellspacing="1" cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td width="304"> | + | <td width="304">▪Miniprep: 22M<br /> |
- | + | ▪Cut: 22M</td> | |
- | <td width="298"> | + | <td width="298">▪Check CFP expression. No fluorescence.</td> |
</tr> | </tr> | ||
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<table width="618" border="0" cellspacing="1" cellpadding="1"> | <table width="618" border="0" cellspacing="1" cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td> | + | <td>▪Transform: 13K, 10I, 22M, 12I, 18P</td> |
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<table width="609" border="0" cellspacing="1" cellpadding="1"> | <table width="609" border="0" cellspacing="1" cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td width="277"> | + | <td width="277">▪Miniprep: 13K, 10I, 22M, 12I, 18P</td> |
- | <td width="227"> | + | <td width="227">▪Cut: 13K, 10I, 22M, 12I, 18P</td> |
- | <td width="95"> | + | <td width="95">▪Run the gel</td> |
</tr> | </tr> | ||
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<table width="620" border="0" cellspacing="1" cellpadding="1"> | <table width="620" border="0" cellspacing="1" cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td> | + | <td>▪Ligate 13K+10I, 22M+10I</td> |
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<table width="620" border="0" cellspacing="1" cellpadding="1"> | <table width="620" border="0" cellspacing="1" cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td width="244"> | + | <td width="244">▪Transform: the Gibson assembly results.</td> |
- | <td width="164"> | + | <td width="164">▪Miniprep: 13K+10I<br /> |
- | + | ▪Cut: 13K+10I</td> | |
- | <td> | + | <td>▪Purification: the digestion results.<br /> |
- | + | ▪Ligation: nirB+13K+10I</td> | |
</tr> | </tr> | ||
</table> | </table> | ||
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cellpadding="1"> | cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td width="191"> | + | <td width="191">▪Transform 13K+10I, 22M+10I</td> |
- | <td width="249"> | + | <td width="249">▪Test new primers.<br /> |
- | + | ▪PCR: 22M+10I, 13K+10I</td> | |
- | <td width="157"> | + | <td width="157">▪Run the PCR products.<br /> |
- | + | ▪Cut the PCR products.</td> | |
</tr> | </tr> | ||
</table> | </table> | ||
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cellpadding="1"> | cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td width="140"> | + | <td width="140">▪Test primers: CS/CP, VF/VR, pSB1_f/r</td> |
- | <td width="181"> | + | <td width="181">▪Cut: 11P, 1F, 22M<br /> |
- | + | ▪Run the cut results.1F-1/2/3 are right.</td> | |
- | <td width="126"> | + | <td width="126">▪Mix the 1F-1/2 purification products</td> |
- | <td width="145"> | + | <td width="145">▪Cut: 1F-1+1F-2(E+S), 22M-3(X+P), pSB1C3(E+P)</td> |
</tr> | </tr> | ||
</table> | </table> | ||
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<table width="594" border="0" cellspacing="1" cellpadding="1"> | <table width="594" border="0" cellspacing="1" cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td width="590"> | + | <td width="590">▪Amplify: vgb, 1C3</td> |
</tr> | </tr> | ||
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<table width="606" border="0" cellspacing="1" cellpadding="1"> | <table width="606" border="0" cellspacing="1" cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td width="602"> | + | <td width="602">▪Digest: Vgb, 1C3, fdfhF, 22M+10I, 13K+10I</td> |
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<table width="608" border="0" cellspacing="1" cellpadding="1"> | <table width="608" border="0" cellspacing="1" cellpadding="1"> | ||
<tr> | <tr> | ||
- | <td width="366"> | + | <td width="366">▪Ligate: vgb+22M+10I, fdfhF+13K+10I</td> |
- | <td width="235"> | + | <td width="235">▪Transform the ligation results.</td> |
</tr> | </tr> | ||
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<h1>1st August</h1> | <h1>1st August</h1> | ||
- | <p>Freeze slicing of about | + | <p>Freeze slicing of about 50μm. Observe under natural light |
- | microscope and can see red florescence. The thickness is about | + | microscope and can see red florescence. The thickness is about 130μm</p> |
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<h1>16th August</h1> | <h1>16th August</h1> | ||
<p>Substituting a part of silicone tube with glass tube in silicone | <p>Substituting a part of silicone tube with glass tube in silicone | ||
- | tube biofilm formation sets. Culture in | + | tube biofilm formation sets. Culture in 37℃</p> |
</div> | </div> | ||
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iGem 2011 Home Page</a> <a href="http://ung.igem.org/Special:Upload/"> | iGem 2011 Home Page</a> <a href="http://ung.igem.org/Special:Upload/"> | ||
Upload Files</a> <a | Upload Files</a> <a | ||
- | href="https://2011.igem.org/wiki/index. | + | href="https://2011.igem.org/wiki/index.phptitle=Template:Zjucss_main&action=edit">Edit |
- | CSS</a> <a href="http://ung.igem.org/ | + | CSS</a> <a href="http://ung.igem.org/Team_Wikisyear=2011"> Team Wikis</a> <a |
- | href="mailto:zjuigem@gmail.com | + | href="mailto:zjuigem@gmail.com"><strong>Contact Us</strong></a></div> |
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</body> | </body> | ||
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Latest revision as of 18:40, 28 October 2011
Lab Notes - August
Biobrick Group
Week5
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Week6
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Week7
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Aug.28th Sunday |
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Biofilm Group
1st August
Freeze slicing of about 50μm. Observe under natural light microscope and can see red florescence. The thickness is about 130μm
4th August
Repeat the last experiment with silicone tube. 6rpm/s results in a flowing speed of 68ml/day
6th August
Similar to the last time. Did not use freeze slicing.
15th August
Cultured E.coli in 5ml LB for 12h.
16th August
Substituting a part of silicone tube with glass tube in silicone tube biofilm formation sets. Culture in 37℃
17th August
Add 50ml LB and culture with circular culture.
18th August
12.00 found one free-flow pump stopped. Cooled for 15 minutes and turned on again. Could be over heated. Can see some bacteria on the bottom of the vessel and tube. When the pump started again, the bacteria was washed away.
19th August
Can observe obvious white biofilm where the silicone tube joins the tube but cannot see red florescence, suspect contamination. No biofilm is observed on the glass tube under microscope.
21st, August
Biofilm formation with large test tubes. Inoculate with 1%
e.coli. Rubber tubes are attached to air pump with filter between air
pump and the tube to prevent entrance of germs. Two sets use MSM
+glucose medium and two sets use LB.
Retry with glass tube biofilm formation set.
24th, August
Terminate biofilm formation, the glass slide left in the drawer
to dry. Can see obvious rod like structure under 400 magnification.
(spheres in MSM+glucose culture) No red florescence is seen. Most of the
surface is covered by single layer cells but some part of it has thick
bump like structure.
No biofilm observed on the glass tube.