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| − | h4 {
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| − | </style>
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| − | </head>
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| − |
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| − | <body>
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| − |
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| − | <p align="center">Click the panels to slide down or up</p>
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| − |
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| − | <h2 align="center">Protocols</h2>
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| − |
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| − | <div class="flip" id="flip1" href ="#panelPCR"> <h4>PCR</h4>
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| − | <div class="panel" id="panelPCR">
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| − |
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| − | <h3>PCR</h3>
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| − |
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| − | <h3>Materials</h3>
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| − | <ul>
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| − | <li>Template DNA</li>
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| − | <li>5X HF Phusion Buffer or 10X HF Taq Buffer</li>
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| − | <li>Molecular Grade Water</li>
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| − | <li>dNTPs</li>
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| − | <li>Primers</li>
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| − | <li>DNA Polymerase (Phusion or Taq)</li>
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| − | </ul>
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| − |
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| − | <h3>Procedure</h3>
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| − |
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| − | <h4>Piece Amplification</h4>
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| − | <ol>
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| − | <li>Create the following mixture:</li>
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| − | <table id="myTable" class="tablesorter">
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| − | <thead>
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| − | <tr>
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| − | <th>Component</th>
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| − | <th>50μl reaction</th>
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| − | </tr>
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| − | </thead>
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| − | <tbody>
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| − | <tr>
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| − |
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| − | <td>Molecular grade H<sub>2</sub>O</td>
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| − | <td>Added first. 32.5μL</td>
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| − | </tr>
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| − | <tr>
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| − | <td>5x HF Buffer</td>
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| − | <td>10μL</td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>10 mM dNTPs</td>
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| − | <td>1.0μL</td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>Primer A (10μM)</td>
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| − | <td>2.5μL</td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>Primer B (10μM)</td>
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| − | <td>2.5μL</td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>Template DNA</td>
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| − | <td>1.0μL</td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>DNA polymerase</td>
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| − | <td>Added last. 0.5μL</td>
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| − |
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| − | </tr>
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| − | </tbody>
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| − | </tbody>
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| − | </table>
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| − | <li>Run the PCR reaction in the Thermalcycler with Thermalcycler Program as follows:</li>
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| − |
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| − | <table id="myTable2" class="tablesorter">
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| − | <thead>
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| − | <tr>
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| − | <th>Cycle Step</th>
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| − | <th>Temp(C)</th>
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| − | <th>Time</th>
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| − | <th>Cycles</th>
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| − | </tr>
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| − | </thead>
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| − | <tbody>
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| − | <tr>
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| − | <td>Initial denaturation</td>
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| − | <td>98</td>
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| − | <td>5 min</td>
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| − | <td>1</td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>Denaturation</td>
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| − | <td>98</td>
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| − | <td>10s</td>
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| − | <td>35</td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>Annealing</td>
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| − | <td>LowerTm+3</td>
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| − | <td>15s</td>
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| − | <td>35</td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>Extension</td>
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| − | <td>72</td>
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| − | <td>45s/kb</td>
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| − | <td>35</td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>Final Extension</td>
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| − | <td>72</td>
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| − | <td>10min</td>
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| − | <td>1</td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>Hold</td>
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| − | <td>4</td>
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| − | <td>Hold</td>
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| − | <td>1</td>
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| − |
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| − | </tr>
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| − |
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| − | </tbody>
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| − |
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| − | </table>
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| − | <table id="myTable" class="tablesorter">
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| − |
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| − | </ol>
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| − |
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| − | <h3>Colony PCR</h3>
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| − |
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| − | <ol>
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| − | <li>Create the following mixture:</li>
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| − |
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| − | <thead>
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| − | <tr>
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| − | <th>Component</th>
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| − | <th>50μl reaction</th>
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| − | </tr>
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| − | </thead>
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| − | <tbody>
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| − | <tr>
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| − |
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| − | <td>Molecular grade H2O</td>
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| − | <td>Added first 33.5ul</td>
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| − | </tr>
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| − | <tr>
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| − | <td>5x Phusion HF Buffer</td>
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| − | <td>10μL</td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>10 mM dNTPs</td>
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| − | <td>1.0μL</td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>Primer A (10μM)</td>
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| − | <td>2.5μL</td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>Primer B (10μM)</td>
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| − | <td>2.5μL</td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>Colony</td>
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| − | <td>1</td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>Phusion DNA polymerase</td>
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| − | <td>Added last. 0.5μL</td>
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| − |
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| − | </tr>
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| − | </tbody>
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| − | </tbody>
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| − | </table>
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| − |
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| − | <li>Run the PCR reaction in the Thermalcycler with Thermalcycler Program as follows:</li>
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| − |
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| − |
| |
| − | <table id="myTable2" class="tablesorter">
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| − | <thead>
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| − | <tr>
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| − | <th>Cycle Step</th>
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| − | <th>Temp(C)</th>
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| − | <th>Time</th>
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| − | <th>Cycles</th>
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| − | </tr>
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| − | </thead>
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| − | <tbody>
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| − | <tr>
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| − | <td>Initial denaturation</td>
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| − | <td>98</td>
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| − | <td>5 min</td>
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| − | <td>1</td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>Denaturation</td>
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| − | <td>98</td>
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| − | <td>10s</td>
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| − | <td>35</td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>Annealing</td>
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| − | <td>LowerTm+3</td>
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| − | <td>15s</td>
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| − | <td>35</td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>Extension</td>
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| − | <td>72</td>
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| − | <td>45s/kb</td>
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| − | <td>35</td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>Final Extension</td>
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| − | <td>72</td>
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| − | <td>10min</td>
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| − | <td>1</td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>Hold</td>
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| − | <td>4</td>
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| − | <td>Hold</td>
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| − | <td>1</td>
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| − |
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| − | </tr>
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| − |
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| − | </tbody>
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| − |
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| − | </table>
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| − |
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| − | </ol>
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| − |
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| − | <br><br>
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| − | <span style='text-align:right'>
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| − | <a href='#'>↑ Close</a>
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| − | </span>
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| − |
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| − | </div>
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| − | </div>
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| − | <p></p>
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| − |
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| − |
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| − | <div class="flip" id="flip1" href ="#panelDig"> <h4>Digestion</h4>
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| − | <div class="panel" id="panelDig">
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| − |
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| − | <h3>Digestion</h3>
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| − |
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| − | <h3>Materials</h3>
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| − | <ul>
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| − | <li>2 LB + Antibiotic plates</li>
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| − | <li>Spreaders or glass beads</li>
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| − | <li>LB Media</li>
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| − | <li>Ligated back bone</li>
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| − | <li>Digested back bone(control)</li>
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| − | </ul>
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| − |
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| − | <h3>Procedure</h3>
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| − | 1. Create the following mixture:
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| − | <table id="myTable3" class="tablesorter">
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| − | <thead>
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| − | <tr>
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| − | <th>Component</th>
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| − | <th>50μl reaction</th>
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| − |
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| − | </tr>
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| − | </thead>
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| − | <tbody>
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| − | <tr>
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| − | <td>Molecular Grade Water</td>
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| − | <td>Calculated. Use to make reaction total volume = 50ul</td>
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| − | </tr>
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| − | <tr>
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| − | <td>Custsmart Buffer(different if using pstI)</td>
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| − | <td>5ul</td>
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| − | </tr>
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| − | <tr>
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| − | <td>DNA</td>
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| − | <td>1ug</td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>Enzyme 1</td>
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| − | <td>.5ul</td>
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| − | </tr>
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| − | <tr>
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| − | <td>Enzyme 2</td>
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| − | <td>0.5ul</td>
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| − |
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| − |
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| − | </tr>
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| − |
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| − |
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| − | </tbody>
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| − |
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| − | </table>
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| − |
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| − | <p>2. Digest your mixture in a water bath at 37C for 60min</p>
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| − |
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| − | <br><br>
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| − | <span style='text-align:right'>
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| − | <a href='#'>↑ Close</a>
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| − | </span>
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| − |
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| − | </div>
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| − | </div>
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| − |
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| − | <p></p>
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| − |
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| − | <div class="flip" id="flip1" href ="#panelLig"> <h4>Ligation</h4>
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| − | <div class="panel" id="panelLig">
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| − |
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| − | <h3>Ligation</h3>
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| − |
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| − | <table id="myTable4" class="tablesorter">
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| − | <thead>
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| − | <tr>
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| − | <th>Component</th>
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| − | <th>20μl reaction</th>
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| − |
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| − | </tr>
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| − | </thead>
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| − | <tbody>
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| − | <p>Ligation Using the “Ligation Template” excel sheet, calculate the amount of each component to combine in a ligation mix for an Insert: Backbone ratio of 4:1</p>
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| − | <tr>
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| − | <td>Molecular Grade Water</td>
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| − | <td>Calculated. Use to make reaction total volume = 20ul</td>
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| − | </tr>
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| − | <tr>
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| − | <td>T4 DNA Ligase Buffer</td>
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| − | <td>2ul</td>
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| − | </tr>
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| − | <tr>
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| − | <td>Insert DNA</td>
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| − | <td></td>
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| − |
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| − | </tr>
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| − | <tr>
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| − | <td>Backbone DNA</td>
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| − | <td></td>
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| − | </tr>
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| − | <tr>
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| − | <td>T4 DNA Ligase</td>
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| − | <td>1ul</td>
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| − |
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| − |
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| − | </tr>
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| − |
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| − |
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| − | </tbody>
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| − |
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| − | </table>
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| − | <p>Incubate at 16C overnight on a heating block or at room temperature for 1 hour.</p>
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| − |
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| − | <br><br>
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| − | <span style='text-align:right'>
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| − | <a href='#'>↑ Close</a>
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| − | </span>
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| − |
| |
| − | </div>
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| − | </div>
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| − |
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| − | <p></p>
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| − |
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| − | <div class="flip" id="flip1" href ="#panelTran"> <h4>Transformation</h4>
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| − | <div class="panel" id="panelTran">
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| − |
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| − | <h3>Transformation</h3>
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| − |
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| − | <h3>Materials</h3>
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| − | <ul>
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| − | <li>2 LB + Antibiotic plates</li>
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| − | <li>Spreaders or glass beads</li>
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| − | <li>LB Media</li>
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| − | <li>Ligated back bone</li>
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| − | <li>Digested back bone(control)</li>
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| − | </ul>
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| − |
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| − | <h3>Procedure</h3>
| |
| − | <ol>
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| − | <li>Set water bath to 42C</li>
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| − | <li>Remove LB+Antibiotic plates from 4C and allow them to come to RT.</li>
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| − | <li>Thaw chemically competent cells on ice. Leave in microcentrifuge tube.</li>
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| − | <li>Add 5μL of ligation mix chemically competent cells.</li>
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| − | <li>Add 5uL of digested back bone to chemical competent cells as a control</li>
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| − | <li>Incubate on ice for 30 min.</li>
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| − | <li>Heat shock cells for 60 sec at 42C without shaking.</li>
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| − | <li>Aseptically (by the fire or in the hood) add 250μL of LB media to the tube (DO NOT ADD ANTIBIOTIC AT THIS STEP). Cap tightly.</li>
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| − | <li>Place tube horizontally in shaker. Incubate at 37oC and 225 rpm for 1 hr.</li>
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| − | <li>In the laminar hood, spread 100uL of transformants onto LB+Antibiotic plates.</li>
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| − | <li>Leave plates in 37C incubator overnight. Store remaining liquid cultures in 4oC.</li>
| |
| − | </ol>
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| − |
| |
| − | <br><br>
| |
| − | <span style='text-align:right'>
| |
| − | <a href='#'>↑ Close</a>
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| − | </span>
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| − |
| |
| − | </div>
| |
| − | </div>
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| − |
| |
| − |
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| − |
| |
| − | <p></p>
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| − |
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| − | <div class="flip" id="flip1" href ="#panel1"> <h4>LB Media and Plates</h4>
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| − | <div class="panel" id="panel1">
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| − | <h3>LB Media</h3>
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| − |
| |
| − | <h3>Materials</h3>
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| − | <ul> <li>LB Miller (Powder)</li>
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| − | <li>1L Glass Bottle</li>
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| − | <li>Stir Bar</li>
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| − | <li>DI/RO Water</li>
| |
| − | </ul>
| |
| − | <h3 >Procedure</h3>
| |
| − | <ol> <li>Triple rinse bottle with DI water</li>
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| − | <li>Add stir bar to the bottle</li>
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| − | <li>Add 700 mL of DI Water to the 1L Bottle</li>
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| − | <li>Add 17.5 g of LB Miller </li>
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| − | <li>Put of stir plate and stir until large clumps are dissolved (Remaining small clumps will dissolve in Autoclave)</li>
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| − | <li>Tighten the cap all the way and loosen with 2 full turns to the left [IMPORTANT for SAFETY]</li>
| |
| − | <li>Place autoclave tape on top</li>
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| − | <li>Autoclave on Liq 30</li>
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| − | <li>Let cool and then tighten the cap and store at room temperature</li>
| |
| − | </ol>
| |
| − |
| |
| − | <h3>LB Agar Media for Plates</h3>
| |
| − | <h3>Materials</h3>
| |
| − | <ul> <li>LB Miller (Powder)</li>
| |
| − | <li>1L Glass Bottle</li>
| |
| − | <li>Stir Bar</li>
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| − | <li>DI/RO Water</li>
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| − | <li>Agar</li>
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| − | <li>Antibiotic if applicable</li>
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| − | <li>Petri Dish Plates</li>
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| − | <li>Ethanol Proof Markers</li>
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| − | <li>Tape</li>
| |
| − | </ul>
| |
| − | <h3 >Procedure</h3>
| |
| − | <ol> <li>Triple rinse bottle with DI water</li>
| |
| − | <li>Add stir bar to the bottle</li>
| |
| − | <li>Add 700 mL of DI Water to the 1L Bottle</li>
| |
| − | <li>Add 17.5 g of LB Miller </li>
| |
| − | <li>Add 8.4g of agar (not agarose)</li>
| |
| − | <li>Put of stir plate and stir until large clumps are dissolved (Remaining small clumps will dissolve in Autoclave)</li>
| |
| − | <li>Tighten the cap all the way and loosen with 2 full turns to the left [IMPORTANT for SAFETY]</li>
| |
| − | <li>Place autoclave tape on top</li>
| |
| − | <li>Autoclave on Liq 30</li>
| |
| − | <li>Remove IMMEDIATELY from autoclave when finished</li>
| |
| − | <li>Place on stir plate and slowly stir (no air bubbles from stirring too fast) until the bottle can be held comfortable for a 7 seconds (20-30 min of cool down time)</li>
| |
| − | <li> Add 700 μL of aliquoted Antibiotic</li>
| |
| − | <li>INSIDE THE HOOD:
| |
| − | <ol>
| |
| − | <li>Spray the sleeve of plates, the agar media, the marker, and the tape down with ethanol before putting them in the hood. When opening the sleeve of plates, be careful to not rip it because you will put the plates back into it.</li>
| |
| − | <li>Following sterile technique pour the plates about a 3rd of the way full</li>
| |
| − | <li>Allow to cool in the hood until they have solidified and are no longer warm</li>
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| − | <li>Turn the plates upside down and put back in the sleeve</li>
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| − | <li>Tape the sleeve shut and write the antibiotic and the date they were made on the tape. </li>
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| − | <h3>Gel Electrophoresis</h3>
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| − | <h3>Materials</h3>
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| − | <ul> <li>Agarose</li>
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| − | <li>1X TAE Buffer</li>
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| − | <li>Parafilm</li>
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| − | <li>Electrophoresis chamber and power source</li>
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| − | <li>SYBR Green</li>
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| − | <li>Loading Dye</li>
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| − | <li>DNA Ladder</li>
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| − | <h3 >Procedure</h3>
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| − | <li>Using a balance, mass out 0.5 gram of agarose (for 1% gel, for 2% mass out 1.0 gram). Mix this with 50 mL of TAE 1X Buffer.</li>
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| − | <li>Microwave the mixture, mixing by swirling intermittently, until all the agarose is dissolved and the mixture is homogeneous and clear (about 2 minutes).</li>
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| − | <li>Pour the mixture into a gel plate and insert a correctly-sized comb (8, 10, etc. lanes). Allow to cool and solidify on the benchtop.</li>
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| − | <li>Mix your samples on a piece of parafilm. Use 10 μL of the appropriate DNA Ladder (1 kb, 100 bp, 2 log, etc.) combined with 1 μL SYBR Green/DMSO in the first lane. For all samples, mix 10 μl of sample and 2 μL of SYBR Green/Dye Mixture.</li>
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| − | <li>Place gel in tray into the gel electrophoresis apparatus - wells should be close to the negative (conventionally black) end. Fill apparatus with 1X TAE until the gel is completely submerged. Do not overfill - there is a guide sticker on the apparatus which marks the max fill line.</li>
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| − | <li>Load 10 μL of each sample into the appropriate wells.</li>
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| − | <li>Connect wiring and run gel electrophoresis at 110 V for 1 hour.</li>
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| − | <li>Analyze gel in UV light and see if samples match the expected size using the ladder as a guide.</li>
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