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| <!-- Page Content --> | | <!-- Page Content --> |
− | <div class="container"> | + | <div class="batu" style="background: url('https://static.igem.org/mediawiki/2017/f/fe/Npu-background.png') no-repeat fixed;"> |
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− | <!-- Page Heading/Breadcrumbs -->
| + | <div class="row"> |
| + | <div class="col-lg-12"> |
| + | <h1 class="page-header">Silver</h1> |
| + | </div> |
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− | <ul class="nav nav-pills nav-stacked" data-spy="affix" style="width:250px; position:fixed;">
| + | |
− | <li class="active"><a href="#section-1">DNA gel extraction</a></li>
| + | |
− | <li><a href="#section-2">Electrophoresis</a></li>
| + | |
− | <li><a href="#section-3">Gibson assenbly</a></li>
| + | |
− | <li><a href="#section-4">HPLC</a></li>
| + | |
− | <li><a href="#section-5">Knock out genes of Ecoli</a></li>
| + | |
− | <li><a href="#section-6">Crispr-cas9</a></li>
| + | |
− | <li><a href="#section-7">Plasmid preparation</a></li>
| + | |
− | <li><a href="#section-8">Plasmid transformation</a></li>
| + | |
− | <li><a href="#section-9">Point mutation</a></li>
| + | |
− | <li><a href="#section-10">Reagents</a></li>
| + | |
− | <li><a href="#section-11">References</a></li>
| + | |
− | <li><a href="#section-12">the LiAc SS carrier DNA PEG method</a></li>
| + | |
− | <li><a href="#section-13">Whole cell catalysis</a></li>
| + | |
− | </ul>
| + | |
− | </div>
| + | |
− | <!-- Content Column -->
| + | |
− | <div class="col-md-9">
| + | |
| | | |
− | <h2 id="section-1" style="padding-top: 100px; margin-top: -50px;">DNA gel extraction</h2>
| + | <img class="img-responsive img-hover" src="http://placehold.it/600x300" alt=""> |
− | <p>
| + | |
− | 1. Excise the agarose gel slice, transfer the gel slice into a 1.5ml microfuge tube.<br /> 2. Add a 3X sample
| + | |
− | volume of Buffer DE-A. the weight of gel is equivalent to a 100ul volume.<br /> 3. Resuspend the gel in Buffer
| + | |
− | DE-A by vortexing. Heat at 75℃ until the gel is completely dissolved(no more than 10 min)<br /> 4. Add 0.5X Buffer
| + | |
− | DE-A volume of Buffer DE-B, mix. <br /> 5. Place a miniprep colume into a 2ml microfuge tube. Transfer the solubilized
| + | |
− | agarose from step 4 into the column. Centrifuge at 12,000xg for 1 min.<br /> 6. Discard the filtrate. Add 500ul
| + | |
− | of Buffer W1. Centrifuge at 12,000xg for 30s.<br /> 7. Discard the filtrate. Add 700ul of Buffer W2. Centrifuge
| + | |
− | at 12,000xg for 30s.<br /> 8. Repeat wash with W2. Centrifuge at 12,000xg for 1min.<br /> 9. Discard the filtrate.
| + | |
− | Centrifuge at 12,000xg for 1min.<br /> 10. Transfer the miniprep column into a clean 1.5ml microfuge tube. Add
| + | |
− | 25-30ul of Eluent or deionized water to the center of the membrane. Let it stand for 1min at room temperature.
| + | |
− | Centrifuge at 12,000xg for 1min. (pre-warming the Eluent at 65℃)<br />
| + | |
− | </p>
| + | |
− | <h2 id="section-2" style="padding-top: 100px; margin-top: -50px;">Electrophoresis</h2>
| + | |
− | <p>Agarose-Electrophoresis is used in order to see if the PCR product is correct and seperate DNA by the number of
| + | |
− | base pairs.
| + | |
− | <br /> ●marker was the 2 kb Plus DNA Ladder<br /> ●fill pockets with 5 µl DNA ladder or 10 µl sample volume with
| + | |
− | 6x loading buffer<br /> ●running conditions: 130 V for 30-40 minutes<br />
| + | |
− | </p>
| + | |
− | <h2 id="section-3" style="padding-top: 100px; margin-top: -50px;">Gibson assembly</h2>
| + | |
− | <p>
| + | |
− | 1. Set up the reaction.<br /> V(ul)=(0.02*bp)/concentration(ng/ul)
| + | |
− | <br /> 2. Add the fragments into Gibson system.<br /> 3. Incubate samples in a thermocycler at 50 °C for 1h.
| + | |
− | <br /> 4. Purify the product using DNA purification kit.<br /> 5. Transform the product into the competent cells
| + | |
− | of E.coli BL21 , following the transformation protocol.<br />
| + | |
− | <br /> Colony pcr system:20µl</p>
| + | |
− | <table border="1" cellspacing="0" cellpadding="0" width="308">
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>Component<br> sterile water</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>Concentration<br> 4µl
| + | |
− | </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>Forward primer</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>1µl</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>Reversed primer</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>1µl</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>Template</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>4µl</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>Mix</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>10µl</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | <p> </p>
| + | |
− | <p>Method:</p>
| + | |
− | <table border="1" cellspacing="0" cellpadding="0" width="484">
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p> </p>
| + | |
− | <p>1 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <p>STEP<br> Initial </p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>TEM<br> 98℃
| + | |
− | </p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>TIME<br> 5min
| + | |
− | </p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p>CYCLES<br> 1
| + | |
− | </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>2 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <ul>
| + | |
− | <li>denaturation</li>
| + | |
− | </ul>
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>98℃</p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>20s</p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>3 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <p>Annealing</p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>58℃</p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>20s </p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>4 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <p>extension</p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>72℃</p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>1min</p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>5 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <ul>
| + | |
− | <li>circulation</li>
| + | |
− | </ul>
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>Repeat the process2-4 </p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p>34 </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>6 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <p>extension</p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>72℃</p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>5min </p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p>1</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>7 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <p>Hold</p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>4℃</p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>Hold</p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | <p> </p>
| + | |
− | <p> </p>
| + | |
− | <h2 id="section-4" style="padding-top: 100px; margin-top: -50px;">HPLC</h2>
| + | |
− | <p>
| + | |
− | for acrylic acid</p>
| + | |
− | <table border="1" cellspacing="0" cellpadding="0">
| + | |
− | <tr>
| + | |
− | <td>
| + | |
− | <p align="center"><strong>Retention time</strong></p>
| + | |
− | </td>
| + | |
− | <td width="265">
| + | |
− | <p align="center"><strong>Lower detection limit</strong></p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td>
| + | |
− | <p>HPLC</p>
| + | |
− | </td>
| + | |
− | <td width="265">
| + | |
− | <p>Waters</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td>
| + | |
− | <p>Column</p>
| + | |
− | </td>
| + | |
− | <td width="265">
| + | |
− | <p>HSS</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td>
| + | |
− | <p>Detector</p>
| + | |
− | </td>
| + | |
− | <td width="265"></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td>
| + | |
− | <p>Column Temperature</p>
| + | |
− | </td>
| + | |
− | <td width="265"></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td>
| + | |
− | <p>Solvent</p>
| + | |
− | </td>
| + | |
− | <td width="265">
| + | |
− | <p>1 mM H2SO4</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td>
| + | |
− | <p>Flow Rate</p>
| + | |
− | </td>
| + | |
− | <td width="265">
| + | |
− | <p>0.6 ml/min</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td>
| + | |
− | <p>Duration</p>
| + | |
− | </td>
| + | |
− | <td width="265">
| + | |
− | <p>30 min</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td>
| + | |
− | <p>Injection Volume</p>
| + | |
− | </td>
| + | |
− | <td width="265">
| + | |
− | <p>10 µl</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | <p><br /> Samples have to be centrifuged at 12,000xg for 5 min in order to remove solids (cells/precipitates).<br
| + | |
− | />
| + | |
− | </p>
| + | |
− | <h2 id="section-5" style="padding-top: 100px; margin-top: -50px;">Knock out genes of E ▪ coli (MG1655)</h2>
| + | |
− | <p>1. Pre-chill 1.5ml and 2ml microcentrifuge tubes, deionized water, 10% glycerol. Add 1ml LB to 2ml microcentrifuge
| + | |
− | tubes.
| + | |
− | <br /> 2. When OD600=0.6, incubate competent cells on ice for 20 min.<br /> 3. Transfer the competent cells to
| + | |
− | 50 mL pre-chilled centrifuge tube. Centrifuge at 5,500r/min, 4 ℃ for 5 min.<br /> 4. Discard the filtrate. Add
| + | |
− | 30ml of pre-chilled deionized water, resuspend the cells gently.<br /> 5. Centrifuge at 5,500r/min, 4 ℃ for 5
| + | |
− | min. Discard the filtrate. Repeat wash with deionized water.<br /> 6. Discard the filtrate. Add 30ml of pre-chilled
| + | |
− | 10% glycerol, resuspend the cells gently. <br /> 7. Centrifuge at 6,500r/min, 4 ℃ for 5 min. Discard the filtrate.
| + | |
− | Repeat wash with 10% glycerol.<br /> 8. Discard the filtrate and leave over 1ml 10% glycerol to resuspend the
| + | |
− | competent cells, pipet 80ul into each 1.5ml EP tube, add 5ul of DNA and carefully mix with the competent cells.
| + | |
− | Let it stand for 2min.<br /> 9. Add electrocompetent to DNA on ice. Move the mixture to the cuvette. Dry and
| + | |
− | shock the cells(2500V). Add 1ml of LB medium. Incubate at 30℃ for 4 hours shaking at 220rpm, pipet 100ul from
| + | |
− | each tube onto the appropriate plate, and spread the mixture evenly across the plate. Incubate at 30℃ overnight.
| + | |
− | Position the plates with the agar side at the top, and the lid at the bottom.<br />
| + | |
− | </p>
| + | |
− | <h2 id="section-6" style="padding-top: 100px; margin-top: -50px;">Knock out the genes of Saccharomyces cerevisiae with Crispr-Cas9</h2>
| + | |
− | <p>
| + | |
− | 1. pcr system:200µl</p>
| + | |
− | <table border="1" cellspacing="0" cellpadding="0" width="308">
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>Component<br> sterile water</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>Concentration<br> 129µl
| + | |
− | </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>5×Fast pfu Buffer </p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>40µl</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>dNTPs</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>16µl</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>Forward primer</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>5µl</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>Reversed primer</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>5µl</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>pCAS_Phe-URA3</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>2µl</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>Fastpfu</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>3µl</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | <p> </p>
| + | |
− | <p>Method:</p>
| + | |
− | <table border="1" cellspacing="0" cellpadding="0" width="484">
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p> </p>
| + | |
− | <p>1 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <p>STEP<br> Initial </p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>TEM<br> 95℃
| + | |
− | </p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>TIME<br> 2min
| + | |
− | </p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p>CYCLES<br> 1
| + | |
− | </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>2 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <ul>
| + | |
− | <li>denaturation</li>
| + | |
− | </ul>
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>95℃</p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>20s</p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>3 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <p>Annealing</p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>58℃</p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>30s </p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>4 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <p>extension</p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>72℃</p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>1min40s </p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>5 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <ul>
| + | |
− | <li>circulation</li>
| + | |
− | </ul>
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>Repeat the process2-4 </p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p>35 </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>6 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <p>extension</p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>72℃</p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>5min </p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p>1</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>7 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <p>Hold</p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>4℃</p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>Hold</p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | <p><br /> 2. Purify the PCR product with a DNA purification kit.<br /> 3. Add the appropriate amount of DMT enzyme,
| + | |
− | hold for one hour at 37 ° C.<br /> 4. transform the DNA into competent cells.<br /> 50ul competent cell + 15ul
| + | |
− | purified DNA,incubate on ice for 30min,heat shock 45s,incubate on ice for 2min,add LB medium and incubate for
| + | |
− | 1h.<br /> 5. Pipet 100ul from each tube onto the plate with resistance, and spread the mixture evenly across
| + | |
− | the plate. Incubate for 12h. Position the plates with the agar side at the top, and the lid at the bottom.<br
| + | |
− | /> 6. use a sterile pipet tip to pick Saccharomyces cerevisiae from plates,throw the tip into the tubes of 5
| + | |
− | ml of LB + antibiotics,incubate in a rotary shaker. Prepare plasmid with kit for sequencing.<br /> 7. Transfer
| + | |
− | plasmid and fragment into Saccharomyces cerevisiae using the LiAc SS carrier DNA PEG method.</p>
| + | |
− | <table border="1" cellspacing="0" cellpadding="0" width="313">
| + | |
− | <tr>
| + | |
− | <td width="162" colspan="2" valign="top">
| + | |
− | <p>plasmid</p>
| + | |
− | <p> </p>
| + | |
− | <p>CRISPR-GPD1_5_0</p>
| + | |
− | </td>
| + | |
− | <td width="150" valign="top">
| + | |
− | <p>fragment(with 1000bp from forward and reverse)<br> GPD1 </p>
| + | |
− | </td>
| + | |
− | <td width="1">
| + | |
− | <p> </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="5">
| + | |
− | <p> </td>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>CRISPR-GPD1_16_0</p>
| + | |
− | </td>
| + | |
− | <td width="151" colspan="2" valign="top">
| + | |
− | <p>GPD1 </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="5">
| + | |
− | <p> </td>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | <td width="151" colspan="2" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="5">
| + | |
− | <p> </td>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>CRISPR-DLD1_11_0</p>
| + | |
− | </td>
| + | |
− | <td width="151" colspan="2" valign="top">
| + | |
− | <p>DLD1 </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="5">
| + | |
− | <p> </td>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>CRISPR-DLD1_18_0</p>
| + | |
− | </td>
| + | |
− | <td width="151" colspan="2" valign="top">
| + | |
− | <p>DLD1 </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="5">
| + | |
− | <p> </td>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>CRISPR-PDC1_6_0<br> CRISPR-PDC1_45_0
| + | |
− | <br> CRISPR-PDC5_13_0
| + | |
− | <br> CRISPR-PDC5_29_0
| + | |
− | </p>
| + | |
− | </td>
| + | |
− | <td width="151" colspan="2" valign="top">
| + | |
− | <p>PDC1<br> PDC1
| + | |
− | <br> PDC5
| + | |
− | <br> PDC5
| + | |
− | </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | <p><br /> 8. Prepare the template: use a sterile toothpick to pick Saccharomyces cerevisiae from plates,put the toothpick
| + | |
− | into 100ul 20mMNaOH and mix,99° C boiling for 30min. Colony pcr system:20µl</p>
| + | |
− | <table border="1" cellspacing="0" cellpadding="0" width="308">
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>Component<br> sterile water</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>Concentration<br> 4µl
| + | |
− | </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>Forward primer</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>1µl</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>Reversed primer</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>1µl</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>Template</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>4µl</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>Mix</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>10µl</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | <p> </p>
| + | |
− | <p>Method:</p>
| + | |
− | <table border="1" cellspacing="0" cellpadding="0" width="484">
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p> </p>
| + | |
− | <p>1 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <p>STEP<br> Initial </p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>TEM<br> 98℃
| + | |
− | </p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>TIME<br> 5min
| + | |
− | </p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p>CYCLES<br> 1
| + | |
− | </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>2 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <ul>
| + | |
− | <li>denaturation</li>
| + | |
− | </ul>
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>98℃</p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>20s</p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>3 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <p>Annealing</p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>58℃</p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>20s </p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>4 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <p>extension</p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>72℃</p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>1min</p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>5 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <ul>
| + | |
− | <li>circulation</li>
| + | |
− | </ul>
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>Repeat the process2-4 </p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p>34</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>6 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <p>extension</p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>72℃</p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>5min </p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p>1</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>7 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <p>Hold</p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>4℃</p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>Hold</p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
| | | |
− | <p> </p>
| + | </div> |
− | <h2 id="section-7" style="padding-top: 100px; margin-top: -50px;">plasmid preparation</h2>
| + | <div class="col-md-6"> |
− | <p>Tiangen mini plasmid kit<br /></p>
| + | <h3> |
| + | Lecture in the school-- To spread iGEM and get more students on board with us! |
| + | </h3> |
| + | <p>December 8, 2016. |
| + | </p> |
| + | <p>Our team arranged a recruitment talk to the students, which appealed to a mountain of students fond of |
| + | Synthetic biology from the School of Astronautics, the school of Automation, and the School of Mechatronics |
| + | and Engineering and so on.</p> |
| | | |
− | <h2 id="section-8" style="padding-top: 100px; margin-top: -50px;">Plasmid transformation</h2>
| + | </div> |
− | <p>1. Pipette 50µl of competent cells and 2µl of plasmid into 1.5ml tube<br /> 2. Heat shock tubes at 42°C for 30s<br
| + | </div> |
− | /> 3. Incubate on ice for 2min<br /> 4. Pipette 250µl LB media to each transformation<br /> 5. Incubate at 37°C | + | <!-- /.row --> |
− | for 1h<br /> 6. Plating<br /> 7. Pick single colonies<br /> Reference: http://parts.igem.org/Help:Protocols/Transformation<br | + | |
− | />
| + | |
− | </p>
| + | |
| | | |
− | <h2 id="section-9" style="padding-top: 100px; margin-top: -50px;">Point mutation</h2>
| + | <hr> |
− | <p>
| + | |
− | 1. pcr system: 50µl</p> | + | |
− | <table border="1" cellspacing="0" cellpadding="0" width="308">
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>Component<br> sterile water</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>Concentration<br> 35µl
| + | |
− | </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>5*TransStart FastPfu buffer </p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>10µl</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>10mM dNTPs</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>1µl</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>10μM Forward primer</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>1µl</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>10μM Reversed primer</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>1µl</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>template</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>1µl</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="157" valign="top">
| + | |
− | <p>TransStart FastPfu DNA polymerase</p>
| + | |
− | </td>
| + | |
− | <td width="151" valign="top">
| + | |
− | <p>1µl</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | <p> </p>
| + | |
− | <p>Method:</p>
| + | |
− | <table border="1" cellspacing="0" cellpadding="0" width="484">
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p> </p>
| + | |
− | <p>1 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <p>STEP<br> Initial </p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>TEM<br> 94℃
| + | |
− | </p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>TIME<br> 5min
| + | |
− | </p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p>CYCLES<br> 1
| + | |
− | </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>2 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <ul>
| + | |
− | <li>denaturation</li>
| + | |
− | </ul>
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>94℃</p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>30s</p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>3 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <p>Annealing</p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>58℃</p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>30s </p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>4 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <p>extension</p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>72℃</p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>3min40s </p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>5 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <ul>
| + | |
− | <li>circulation</li>
| + | |
− | </ul>
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>Repeat the process2-4 </p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p>30 </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>6 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <p>extension</p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>72℃</p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>10min </p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p> 1</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="25" valign="top">
| + | |
− | <p>7 </p>
| + | |
− | </td>
| + | |
− | <td width="113" valign="top">
| + | |
− | <p>Hold</p>
| + | |
− | </td>
| + | |
− | <td width="103" valign="top">
| + | |
− | <p>4℃</p>
| + | |
− | </td>
| + | |
− | <td width="102" valign="top">
| + | |
− | <p>Hold</p>
| + | |
− | </td>
| + | |
− | <td width="142" valign="top">
| + | |
− | <p> </p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | <p><br /> 2. Purify the PCR product with a DNA purification kit.<br /> 3. Add the appropriate amount of DMT enzyme,
| + | |
− | hold for one hour at 37 ° C.<br /> 4. Transform 5μl digested DNA into competent cells DH5α, incubate on ice for
| + | |
− | 30min.
| + | |
− | <br /> 42° C heat shock, 45s. Incubate on ice for 2min.<br /> add 200μl of LB. incubate at 37 °C for 1 h, 220rpm/min.
| + | |
− | <br /> 5. pipet 200μl from each tube onto the plate with appropriate resistance, and spread the mixture evenly
| + | |
− | across the plate. Incubate at 37℃ overnight. Position the plates with the agar side at the top, and the lid at
| + | |
− | the bottom.<br /> 6. Select single colonies for sequencing.<br />
| + | |
− | </p>
| + | |
| | | |
− | <h2 id="section-10" style="padding-top: 100px; margin-top: -50px;">Reagents</h2>
| + | <!-- Blog Post Row --> |
− | <p>1. LB medium(lysogeny broth)<br /> The recipe for 1l LB media is as follows:<br /> Tryptone 10g/L <br /> Yeast
| + | <div class="row"> |
− | extract 5g/L <br /> NaCl 10g/L<br /> 2. 0.1mM Kanamycin<br /> MW of Kanamycin:582.58<br /> Store at -20℃<br /> 3. LB plate<br /> The recipe for 1l LB plate is as follows:<br /> Tryptone 10g/L <br /> Yeast extract 5g/L <br | + | |
− | /> NaCl 10g/L<br /> 15g Agar<br /> Add appropriate amount of resistance.<br /> 4. 2YT medium<br /> The recipe
| + | |
− | for 1l LB plate is as follows:<br /> Tryptone 16g/L <br /> Yeast extract 10g/L <br /> NaCl 5g/L<br /> 5. 0.5mM
| + | |
− | IPTG <br /> MW of IPTG:238.30<br /> Store at -20℃<br /> 6. 50mM PBS buffer,PH8.0<br /> A:0.05mol/L Na2HPO4溶液<br
| + | |
− | /> B:0.05mol/L KH2P04溶液<br /> 137mMNaCl,2.7mMKCl,10mMNa2HPO4,2mMKH2PO4 for 1L.<br />
| + | |
| | | |
− | </p>
| + | <div class="col-md-6"> |
| | | |
− | <h2 id="section-11" style="padding-top: 100px; margin-top: -50px;">References</h2>
| + | <img class="img-responsive img-hover" src="http://placehold.it/600x300" alt=""> |
− | <p>http://parts.igem.org/Help:Protocols/Transformation<br /> https://2015.igem.org/Team:Aachen/Project/Overview
| + | |
− | <br /> http://www.zymoresearch.com/category/all-products
| + | |
− | <br /> http://www.corning.com/worldwide/en/products/life-sciences/resources/brands/axygen-brand-products.html
| + | |
− | <br /> http://www.tsingke.net/shop/
| + | |
− | <br /> http://www.cwbiotech.com/
| + | |
− | <br />
| + | |
− | </p>
| + | |
| | | |
− | <h2 id="section-12" style="padding-top: 100px; margin-top: -50px;">the LiAc SS carrier DNA PEG method</h2>
| + | </div> |
− | <p>
| + | <div class="col-md-6"> |
− | 1.use a sterile pipet tip to pick Saccharomyces cerevisiae from plates,throw the tip into the tubes of appropriate medium,incubate
| + | <h3>Laboratory---To offer more students access to the biology laboratory! |
− | in a rotary shaker for 12h.<br /> 2.measure OD600, transfer x(x=(50×0.2)/(OD600×dilution ratio))
| + | </h3> |
− | ml Saccharomyces cerevisiae into 50ml YPAD.<br /> 3.incubate for 4-5h to make OD600 reaches 0.8-0.9.<br /> 4.boil
| + | <p>December 11, 2016. |
− | ssDNA.<br /> 5.Centrifuge at 3000g for 5 min. Discard the filtrate. Repeat washes with 25ml deionized water twice.<br
| + | </p> |
− | /> 6.Transfer the cells to 1.5 mL centrifuge tube. Add 1ml of deionized water, resuspend the cells gently.<br
| + | <p>We organized nearly 40 students to visit our lab and introduce all sorts of instruments to them at length. |
− | /> 7.Centrifuge at 13000rpm for 30s. Discard the filtrate.<br /> 8.Add 1ml of deionized water, resuspend the
| + | The students are differentiated by various majors--Automation Engineering, Electronic Information |
− | cells. Pipet 100ul into each 1.5 mL centrifuge tube.<br /> 9.Centrifuge using a Mini Centrifuge. Discard the
| + | Science and Technology, Physics, Chemistry and so on. The participation of students from distinct |
− | filtrate.<br /> 10.System for transformation:</p>
| + | majors, we reckon, can certainly promote more brilliant ideas. There is no escaping the fact that |
− | <table border="1" cellspacing="0" cellpadding="0">
| + | students from other majors, such as Chemistry and Electronic Information, constitute a large proportion |
− | <tr>
| + | of our team rather than those majoring in Biology, and their strong interest in biology accounts |
− | <td width="274" valign="top">
| + | for their choice of joining us and ceaselessly brought surprise to the team utilizing their expertise, |
− | <p>PEG3350(50%)</p>
| + | like our Hardware this year.</p> |
− | </td>
| + | |
− | <td width="271" valign="top">
| + | |
− | <p>240ul</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="274" valign="top">
| + | |
− | <p>LiAc 1.0M</p>
| + | |
− | </td>
| + | |
− | <td width="271" valign="top">
| + | |
− | <p>36 ul</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="274" valign="top">
| + | |
− | <p>SSDNA(2.0mg/ml)</p>
| + | |
− | </td>
| + | |
− | <td width="271" valign="top">
| + | |
− | <p>50 ul</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="274" valign="top">
| + | |
− | <p>DNA and H2O</p>
| + | |
− | </td>
| + | |
− | <td width="271" valign="top">
| + | |
− | <p>34 ul</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="274" valign="top">
| + | |
− | <p>Total</p>
| + | |
− | </td>
| + | |
− | <td width="271" valign="top">
| + | |
− | <p>360ul</p>
| + | |
− | </td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | <p><br /> 11.Incubate at 30℃ for 20min.<br /> 12.42℃ heat shock for 40min. pipet 100ul from each tube onto the appropriate
| + | |
− | plate, and spread the mixture evenly across the plate. Incubate at 30℃ for 2-3 days. Position the plates with
| + | |
− | the agar side at the top, and the lid at the bottom.<br /> 13.Prepare plasmid for sequencing.<br />
| + | |
| | | |
− | </p>
| + | </div> |
| + | </div> |
| + | <!-- /.row --> |
| | | |
− | <h2 id="section-13" style="padding-top: 100px; margin-top: -50px;">Whole-cell catalysis</h2>
| + | <hr> |
− | <p>
| + | |
− | 1. Prepare sterile tubes of 5 ml of 2YT+antibiotics. Use a sterile pipet tip to pick bacteria from plates. Throw the tip | + | |
− | into the tubes. Incubate in a rotary shaker at37℃ for 3-4h.<br /> 2. Transfer 120µl of bacteria from
| + | |
− | a slant culture into an Erlenmeyer flask containing 60 mL LB medium with appropriate resistance, incubate at
| + | |
− | 37 °C.
| + | |
− | <br /> 3. When the OD600 reaches 0.6-0.8, the induction of IPTG (0.5 mM) should be carried out. Incubate at 30
| + | |
− | °C on a rotary shaker incubator at 220 rpm for 14 h.<br /> 4. Harvest the bacteria(6000rpm/min 7min). Wash with
| + | |
− | 30ml PBS.<br /> 5. The biocatalytic reaction mixture contained 10% glycerol, E▪ coli and 50mM PBS. Reaction time
| + | |
− | gradient: 8h, 16h, 32h.<br /> 6. Use HPLC for further analysis.<br />
| + | |
− | <br /> Reference: Li N, He Y, Chen Y, et al. Production of cyclic adenosine-3′,5′-monophosphate by whole cell
| + | |
− | catalysis using recombinant Escherichia coli, overexpressing adenylate cyclase[J]. Korean Journal of Chemical
| + | |
− | Engineering, 2013, 30(4):913-917.<br />
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| + | <h3>Communication---To make us more disciplined and efficient |
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| + | <p>December 20, 2016. |
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| + | <p>We had a tea party with the teachers from the School of Life Sciences, during which we expanded upon |
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| + | <h3>To familiarize more students with iGEM |
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| + | <p>June 3, 2017. |
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| + | <p>We participated in the Forum on Life Science of Shaanxi University League, where we introduced our project |
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