Team:WHU-China/Safety

From 2011.igem.org

(Difference between revisions)
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<h3><a href="#">1st Question</a></h3>
<h3><a href="#">1st Question</a></h3>
<div>
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<p class=MsoListParagraph style='margin-left:36.0pt;text-indent:-36.0pt'><span
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<p>
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lang=EN-US style='font-size:15.0pt;font-family:"Times New Roman","serif"'>1.1<span
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Sed non urna. Donec et ante. Phasellus eu ligula. Vestibulum sit amet
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style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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purus. Vivamus hendrerit, dolor at aliquet laoreet, mauris turpis porttitor
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</span></span><span lang=EN-US style='font-size:15.0pt;font-family:"Times New Roman","serif"'>Storage</span></p>
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velit, faucibus interdum tellus libero ac justo. Vivamus non quam. In
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suscipit faucibus urna.
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<p class=MsoListParagraph style='margin-left:21.0pt;text-indent:-21.0pt;
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</p>
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line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-US style='font-family:"Times New Roman","serif"'>D</span><span
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lang=EN-US style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>ried
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DNA: room temperature</span></p>
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<p class=MsoListParagraph style='margin-left:21.0pt;text-indent:-21.0pt;
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line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>Resuspended DNA: -20</span><span
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style='font-size:12.0pt;line-height:150%;font-family:宋体'>℃</span><span
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lang=EN-US style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>
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freezer</span></p>
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<p class=MsoListParagraph style='margin-left:21.0pt;text-indent:-21.0pt;
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line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>The linearized plasmid backbones
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(25ng/ul at 50ul) should be stored at 4C or lower</span></p>
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<p class=MsoListParagraph style='margin-left:36.0pt;text-indent:-36.0pt'><span
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lang=EN-US style='font-size:15.0pt;font-family:"Times New Roman","serif"'>1.2<span
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style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-US style='font-size:15.0pt;font-family:"Times New Roman","serif"'>Usage</span></p>
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<p class=MsoListParagraph style='margin-left:21.0pt;text-indent:-21.0pt;
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line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>With a pipette tip, punch a hole through
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the foil cover into the corresponding well to the Biobrick&#8482;-standard part that
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you want. </span><span lang=EN-US><a
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href="http://partsregistry.org/Help:Spring_2010_DNA_distribution#Distribution_plate_orientation"
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title="http://partsregistry.org/Help:Spring_2010_DNA_distribution#Distribution_plate_orientation"><span
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style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif";
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color:windowtext;text-decoration:none'>Make sure you have properly oriented the
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plate</span></a></span><span lang=EN-US style='font-size:12.0pt;line-height:
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150%;font-family:"Times New Roman","serif"'>. We recommend that you do not
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remove the foil cover, as it could lead to cross contamination between the
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wells. </span></p>
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<p class=MsoListParagraph style='margin-left:21.0pt;text-indent:-21.0pt;
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line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-US><a
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href="http://partsregistry.org/Image:IGEM06DistPlateTop.jpg"
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title="&quot;Top view of plates containing dry DNA; red circle indicates well 13H&quot; "><span
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style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif";
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color:windowtext;text-decoration:none'><img border=0 width=200 height=134
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id="图片 9" src="/wiki/images/5/5d/Whu-Proto1.jpg"
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alt="说明: Top view of plates containing dry DNA; red circle indicates well 13H"></span></a></span></p>
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<p class=MsoListParagraph style='margin-left:21.0pt;text-indent:-21.0pt;
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line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>Add 10uL of diH2O (deionized water), the
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resuspension will become red due to the cresol red dye used during
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manufacturing. </span></p>
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<p class=MsoListParagraph style='margin-left:21.0pt;text-indent:-21.0pt;
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line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>Pipette 1 or 2uL of the resuspended DNA </span><span
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lang=EN-US><a href="http://partsregistry.org/Help:Transformation_Protocol"
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title="Help:Transformation Protocol"><span style='font-size:12.0pt;line-height:
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150%;font-family:"Times New Roman","serif";color:windowtext;text-decoration:
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none'>transform</span></a></span><span lang=EN-US style='font-size:12.0pt;
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line-height:150%;font-family:"Times New Roman","serif"'> into your desired
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competent cells, plate bacteria with the appropriate antibiotic* and grow
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overnight. </span></p>
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<p class=MsoListParagraph style='margin-left:21.0pt;text-indent:-21.0pt;
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line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>5.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>Pick a single bacterial colony and
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transfer it into LB medium. Incubate the culture for 18 hours with vigorous
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agitation.</span></p>
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<p class=MsoListParagraph style='margin-left:21.0pt;text-indent:-21.0pt;
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line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>6.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>Store the single colony in physiological
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saline and on inclined plane, and make recognizable marks on the tube and on
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the notebook.</span></p>
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<a name="poc"><a href="#top" title="Back to top">
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<p class=MsoNormal style='line-height:150%'><span style='font-size:20.0pt;
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line-height:150%;font-family:宋体'>Ⅱ</span><span lang=EN-US style='font-size:
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20.0pt;line-height:150%;font-family:"Times New Roman","serif"'>. Preparation of
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competent cells (Using Calcium Chloride)</span></p></a></a>
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<p class=MsoNormal><span lang=EN-US style='font-size:15.0pt;font-family:"Times New Roman","serif"'>2.1&nbsp;
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Day 0: </span></p>
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<p class=MsoNormal style='line-height:150%'><span lang=EN-US style='font-size:
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12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>&nbsp;&nbsp;&nbsp;
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</span><span lang=EN-US style='font-size:12.0pt;line-height:150%;font-family:
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"Times New Roman","serif"'>Pick a single bacterial colony from a plate that has
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been incubated for 16-20 hours at 37</span><span style='font-size:12.0pt;
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line-height:150%;font-family:宋体'>℃</span><span lang=EN-US style='font-size:
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12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>. Transfer the
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colony into LB medium. Incubate the culture overnight (about 16 hours) at 37</span><span
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style='font-size:12.0pt;line-height:150%;font-family:宋体'>℃</span><span
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lang=EN-US style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>
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with vigorous shaking.</span></p>
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<p class=MsoNormal><span lang=EN-US style='font-size:15.0pt;font-family:"Times New Roman","serif"'>2.2&nbsp;
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Day 1: </span></p>
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<p class=MsoListParagraph style='margin-left:21.0pt;text-indent:-21.0pt;
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line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>Transfer 1 ml of the culture into 100 ml
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of LB medium. Incubate the culture for 2.5-3 hours at 37</span><span
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style='font-size:12.0pt;line-height:150%;font-family:宋体'>℃</span><span
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lang=EN-US style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>
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with vigorous agitation (250-300rpm).</span></p>
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<p class=MsoListParagraph style='margin-left:21.0pt;text-indent:-21.0pt;
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line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>Transfer 1.5 ml of the culture into
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sterile ice-cold 1.5ml polypropylene tubes. Cool the cultures by storing the
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tubes on ice for 10 minutes.</span></p>
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<p class=MsoListParagraph style='margin-left:21.0pt;text-indent:-21.0pt;
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line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>Recover the cells by centrifugation at 2500
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rpm for 5minutes at 4</span><span style='font-size:12.0pt;line-height:150%;
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font-family:宋体'>℃</span><span lang=EN-US style='font-size:12.0pt;line-height:
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150%;font-family:"Times New Roman","serif"'>.</span></p>
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<p class=MsoListParagraph style='margin-left:21.0pt;text-indent:-21.0pt;
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line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>Decant the medium from the cell pellets.
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Resuspend each pellet by swirling or gentle vortexing in 100ml of ice-cold 0.1M
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CaCl2 solution. Store the tubes on ice for 20minutes.</span></p>
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<p class=MsoListParagraph style='margin-left:21.0pt;text-indent:-21.0pt;
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line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>5.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>Recover the cells by centrifugation at
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2500 rpm for 5 minutes at 4</span><span style='font-size:12.0pt;line-height:
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150%;font-family:宋体'>℃</span><span lang=EN-US style='font-size:12.0pt;
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line-height:150%;font-family:"Times New Roman","serif"'>.</span></p>
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<p class=MsoListParagraph style='margin-left:21.0pt;text-indent:-21.0pt;
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line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>6.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>Decant the medium from the cell pellets.
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Resuspend each pellet by swirling or gentle vortexing in 100ml of ice-cold 0.1M
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CaCl2 solution.</span></p>
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<p class=MsoListParagraph style='margin-left:21.0pt;text-indent:-21.0pt;
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line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>7.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>The suspension of competent cells should
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be immediately used in transformation.</span></p>
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<p class=MsoNormal align=left style='margin-bottom:9.6pt;text-align:left'><b><span
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lang=EN-US style='font-size:12.0pt;font-family:"Times New Roman","serif"'>&nbsp;</span></b></p>
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<p class=MsoNormal align=left style='margin-bottom:9.6pt;text-align:left'><b><span
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lang=EN-US style='font-size:12.0pt;font-family:"Times New Roman","serif";
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color:red'>Attention:</span></b></p>
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<p class=MsoListParagraph style='margin-left:21.0pt;text-indent:-21.0pt;
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line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>[1]<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>Mix gently </span></p>
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<p class=MsoListParagraph style='margin-left:21.0pt;text-indent:-21.0pt;
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line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>[2]<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>The newly prepared competent cells
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should be transformed immediately. They cannot be stored for long (except in
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-80</span><span style='font-size:12.0pt;line-height:150%;font-family:宋体'>℃</span><span
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lang=EN-US style='font-size:12.0pt;line-height:150%;font-family:"Times New Roman","serif"'>)</span></p>
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<p class=MsoListParagraph style='margin-left:21.0pt;text-indent:-21.0pt;
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line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>[3]<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>Keep sterile environment during
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operation (sterilize the clean bench by ultra-violet, sterilize hands and implements
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by alcohol). </span></p>
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<p class=MsoListParagraph style='margin-left:21.0pt;text-indent:-21.0pt;
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line-height:150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>[4]<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-US style='font-size:12.0pt;line-height:150%;
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font-family:"Times New Roman","serif"'>Use heat-shock method to transform. </span></p>
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<p class=MsoListParagraph style='margin-left:21.0pt;text-indent:0cm;line-height:
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150%'><span lang=EN-US style='font-size:12.0pt;line-height:150%;font-family:
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"Times New Roman","serif"'>&nbsp;</span></p>
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</div>
</div>
<h3><a href="#">2rd Question</a></h3>
<h3><a href="#">2rd Question</a></h3>

Revision as of 14:24, 26 September 2011

1st Question

Sed non urna. Donec et ante. Phasellus eu ligula. Vestibulum sit amet purus. Vivamus hendrerit, dolor at aliquet laoreet, mauris turpis porttitor velit, faucibus interdum tellus libero ac justo. Vivamus non quam. In suscipit faucibus urna.

2rd Question

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3rd Question

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4th Question

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Suspendisse eu nisl. Nullam ut libero. Integer dignissim consequat lectus. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos.

5th Question

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6th Question

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7th Question

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