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− | <title>iGEM</title>
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− | <ul>
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− | <li><a href="https://2015.igem.org/">iGEM</a></li>
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− | <li><a href="https://2015.igem.org/Teams:Lethbridge">RNAiCare</a></li>
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− | <li>
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− | <a href="https://2015.igem.org/Teams:Lethbridge/Project">Project</a>
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− | <ul>
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− | <li><a href="https://2015.igem.org/Teams:Lethbridge/Description">Description</a></li>
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− | <li><a href="https://2015.igem.org/Teams:Lethbridge/Design">Design</a></li>
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− | <li><a href="https://2015.igem.org/Teams:Lethbridge/Results">Results</a></li>
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− | <li><a href="https://2015.igem.org/Teams:Lethbridge/Project_Production">Production</a></li>
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− | <li><a href="https://2015.igem.org/Teams:Lethbridge/Project_Judging"></a></li>
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− | <a href="https://2015.igem.org/Teams:Lethbridge/Parts">Parts</a>
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− | <ul>
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− | <li><a href="Basic_Part.html">Basic Parts</a></li>
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− | <li><a href="Composite_Part.html">Composite Parts</a></li>
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− | <li><a href="https://2015.igem.org/Teams:Lethbridge/Measurement">Measurement</a></li>
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− | </li>
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− | <a href="https://2015.igem.org/Teams:Lethbridge/Practices">Practices</a>
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− | <ul>
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− | <li><a href="https://2015.igem.org/Teams:Lethbridge/Practices_Risks">Risks</a></li>
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− | <li><a href="https://2015.igem.org/Teams:Lethbridge/Practices_Stakeholders">Stakeholders</a></li>
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− | <li><a href="https://2015.igem.org/Teams:Lethbridge/Practices_Current">Current Problems</a></li>
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− | </ul>
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− | </li>
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− | <a href="https://2015.igem.org/Teams:Lethbridge/Notebook">Notebook</a>
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− | <ul>
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− | <li><a href="https://2015.igem.org/Teams:Lethbridge/Notebook_July">July</a></li>
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− | <li><a href="https://2015.igem.org/Teams:Lethbridge/Notebook_August">August</a></li>
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− | <li><a href="https://2015.igem.org/Teams:Lethbridge/Notebook_September">September</a></li>
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− | </ul>
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− | <a href="https://2015.igem.org/Teams:Lethbridge/Software">Software</a>
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− | <li>
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− | <a href="https://2015.igem.org/Teams:Lethbridge/Team">Team</a>
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− | <ul>
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− | <li><a href="https://2015.igem.org/Teams:Lethbridge/Team_Members">Members</a></li>
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− | <li><a href="https://2015.igem.org/Teams:Lethbridge/Collaborations">Collaborations</a></li>
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− | <li><a href="https://2015.igem.org/Teams:Lethbridge/Attributions">Attributions</a></li>
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− | <li><a href="https://2015.igem.org/Teams:Lethbridge/Sponsors">Sponsors</a></li>
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− | </ul>
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− | </li>
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− | </ul>
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− | </div>
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− | <div id="content_wrapper">
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− | <div class="mini_banner">
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− | </div>
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− | <div class="content_box">
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− | <!-- Notebook Entry -->
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− | <div class="notebook_entry">
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− | <div class="entry_day">
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− | <div>3</div>
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− | </div>
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− | <div class="entry_content">
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− | <div class="entry_title">
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− | <h1>Digestion</h1>
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− | </div>
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− | <div class="entry_data">
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− | <h2>Digest of Device 1 Plasmids and Subsequent Agarose Gel Electrophoresis</h2>
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− | <table>
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− | <tr> <th>Component</th> <th>Volume (μL)</th> </tr>
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− | <tr> <td>Plasmid DNA</td> <td>5</td> </tr>
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− | <tr> <td>10x FD green buffer</td> <td>1</td> </tr>
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− | <tr> <td>FD EcoRI</td> <td>0.5</td> </tr>
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− | <tr> <td>dH2O</td> <td>3.5</td> </tr>
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− | </table>
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− | <p>Total volume: 10μL</p>
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− | </div>
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− | <div class="notebook_entry">
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− | <div class="entry_day">
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− | <div>4</div>
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− | </div>
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− | <div class="entry_content">
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− | <div class="entry_title">
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− | <h1>Miniprep & PCR</h1>
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− | </div>
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− | <div class="entry_data">
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− | <h2>Miniprep Colonies 1 to 5</h2>
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− | <p>Part is TP ribozyme test #1 currently in Pjet1.2 blunt cloning vector. Will bio drop minipreps and PCR amplify out the TP ribozyme test #1 using BB prefix/suffix primers. Spin down 4 mL LB at 13,300 rpm for 2 minutes. Cultures were taken at 3:30 am to be miniprepped.</p>
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− | <ul>
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− | <li>100μL solution I pipette up and down</li>
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− | <li>200μL solution II mix by inverting</li>
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− | <li>300μL solution III mix by inverting</li>
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− | </ul>
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− | <p>Spin at 13,300 for 5 minutes</p>
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− | <p>Pour supernatant onto column</p>
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− | <p>Rinse 2x with wash solution</p>
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− | <p>2x elute in 50μL using the eluta in elution #2</p>
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− | <h2>Bio Drop</h2>
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− | <table>
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− | <tr> <th>Colony</th> <th>Concentration (ng/μL)</th> </tr>
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− | <tr> <td>1</td> <td>204.2</td> </tr>
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− | <tr> <td>2</td> <td>237.2</td> </tr>
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− | <tr> <td>3</td> <td>51.24</td> </tr>
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− | <tr> <td>4</td> <td>66.6</td> </tr>
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− | <tr> <td>5</td> <td>150.9</td> </tr>
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− | </table>
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− | <h2>PCR</h2>
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− | <table>
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− | <tr> <th>Component</th> <th>Volume (μL)</th> <th>Master Mix (x5 reactions)</th> </tr>
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− | <tr> <td>MilliQ H2O</td> <td>39</td> <td>195</td> </tr>
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− | <tr> <td>10x Pfu buffer</td> <td>5</td> <td>25</td> </tr>
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− | <tr> <td>10 nM dNTPs</td> <td>2</td> <td>10</td> </tr>
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− | <tr> <td>BB prefix/suffix primer (10μM)</td> <td>1</td> <td>2</td> </tr>
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− | <tr> <td>Template DNA</td> <td>1</td> <td>-</td> </tr>
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− | <tr> <td>Pfu polymerase (Thermo Sci)</td> <td>1</td> <td>5</td> </tr>
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− | </table>
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− | <p>Total volume: 50μL</p>
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− | <h2>Thermo Cycler</h2>
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− | <table>
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− | <tr> <th>Temperature (°C)</th> <th>Time (seconds)</th> </tr>
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− | <tr> <td>98</td> <td>180</td> </tr>
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− | <tr> <td>98</td> <td>30</td> </tr>
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− | <tr> <td>53</td> <td>30</td> </tr>
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− | <tr> <td>72</td> <td>30</td> </tr>
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− | <tr> <td>72</td> <td>600</td> </tr>
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− | </table>
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− | <p>Note: All 30 seconds runs were repeated 35 times.</p>
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− | </div>
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− | <div class="notebook_entry">
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− | <div class="entry_day">
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− | <div>6</div>
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− | </div>
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− | <div class="entry_content">
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− | <div class="entry_title">
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− | <h1>Miniprep</h1>
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− | </div>
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− | <div class="entry_data">
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− | <h2>Miniprep of Device 2</h2>
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− | <p>12% native PAGE</p>
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− | <ul>
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− | <li>1.8 mL polyacrylamide (40-1)</li>
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− | <li>1.2 mL TBE (5x)</li>
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− | <li>Fill to 6 mL with dH2O</li>
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− | <li>Put in over to help dissolve. Vortex.</li>
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− | <li>Add 10μL APS. Invert</li>
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− | <li>Add 12μL TEMED. Vortex.</li>
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− | </ul>
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− | <p>12% 8M Urea PAGE</p>
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− | <ul>
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− | <li>2.88g urea</li>
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− | <li>1.8μL polyacrylamide</li>
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− | <li>1.2μL TBE (x5)</li>
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− | <li>Fill to 6 mL with dH2O</li>
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− | <li>Add 40μL APS. Invert</li>
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− | <li>Add 6μL TEMED. Vortex</li>
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− | </ul>
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− | </div>
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− | More ↓
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− | </div>
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− | <!-- Notebook Entry -->
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− | <div class="notebook_entry">
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− | <div class="entry_day">
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− | <div>10</div>
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− | </div>
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− | <div class="entry_content">
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− | <div class="entry_title">
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− | <h1>Miniprep</h1>
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− | </div>
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− | <div class="entry_data">
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− | <p>Sending device 1 for sequencing</p>
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− | <ol>
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− | <li>Rep Device 1 col 2 - BB prefix</li>
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− | <li>Rep Device 1 col 2 - BB suffix</li>
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− | <li>Device 1 - BB prefix</li>
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− | <li>Device 1 - BB suffix</li>
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− | </oL>
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− | <ol>
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− | <li>I3504 in pSBIA2 – gel extracted (1% agarose gel)
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− | <ul>
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− | <li>5μL 10x cutsmart buffer</li>
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− | <li>10μL I3504 minipreps</li>
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− | <li>28μL MilliQ H2O</li>
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− | <li>1μL XbaI (20U/μL)</li>
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− | <li>1μL PstI (20U/μL)</li>
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− | </li>
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− | <li>J23117 in pSBIC3
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− | <ul>
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− | <li>5μL 10x cutsmart buffer</li>
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− | <li>15μL miniprep DNA</li>
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− | <li>28μL MilliQ H2O</li>
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− | <li>1μL SpeI</li>
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− | <li>1μL PstI</li>
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− | </il>
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− | </li>
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− | <li>J23106 in pSBIC3
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− | <ul>
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− | <li>5μL 10x cutsmart buffer</li>
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− | <li>15μL miniprep DNA</li>
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− | <li>28μL MilliQ H2O</li>
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− | <li>1μL SpeI</li>
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− | <li>1μL PstI</li>
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− | </ul>
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− | </li>
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− | </ol>
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− | <p>Elute in 35μL water after clean-up</p>
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− | <h2>Ligation Overnight</h2>
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− | <p>Device I → J23101 + I13504</p>
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− | <table>
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− | <tr> <th>Component</th> <th>Volume (μL)</th> </tr>
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− | <tr> <td>T4 DNA ligase buffer</td> <td>1</td> </tr>
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− | <tr> <td>T4 DNA ligase</td> <td>0.5</td> </tr>
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− | <tr> <td>I13504 (gel extracted)</td> <td>2.5</td> </tr>
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− | <tr> <td>J23101 backbone</td> <td>1</td> </tr>
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− | <tr> <td>dH2O</td> <td>5</td> </tr>
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− | </table>
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− | <p>Total volume: 10μL</p>
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− | <p>Device II → J23106 + I13504</p>
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− | <table>
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− | <tr> <th>Component</th> <th>Volume (μL)</th> </tr>
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− | <tr> <td>T4 DNA ligase buffer</td> <td>1</td> </tr>
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− | <tr> <td>T4 DNA ligase</td> <td>0.5</td> </tr>
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− | <tr> <td>I13504</td> <td>2.5</td> </tr>
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− | <tr> <td>J23106 backbone</td> <td>1</td> </tr>
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− | <tr> <td>dH2O</td> <td>5</td> </tr>
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− | </table>
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− | <p>Total volume: 10μL</p>
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− | <p>Device III → J23117 + I13504</p>
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− | <table>
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− | <tr> <th>Component</th> <th>Volume (μL)</th> </tr>
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− | <tr> <td>T4 DNA ligase buffer</td> <td>1</td> </tr>
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− | <tr> <td>T4 DNA ligase</td> <td>0.5</td> </tr>
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− | <tr> <td>I13504</td> <td>2.5</td> </tr>
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− | <tr> <td>J23117 backbone</td> <td>1</td> </tr>
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− | <tr> <td>dH2O</td> <td>5</td> </tr>
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− | </table>
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− | <p>Total volume: 10μL</p>
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− | </div>
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− | <div class="entry_expand_button">
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− | More ↓
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− | </div>
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− | </div>
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− | </div>
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− | <!-- End Notebook Entry -->
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− | <!-- Notebook Entry -->
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− | <div class="notebook_entry">
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− | <div class="entry_day">
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− | <div>11</div>
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− | </div>
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− | <div class="entry_content">
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− | <div class="entry_title">
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− | <h1>In Vitro Transcription</h1>
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− | </div>
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− | <div class="entry_data">
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− | <p>Clone Tt ribozyme test 1 into pJET again. Test with PCR and subsequent in vitro transcription</p>
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− | <table>
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− | <tr> <th>Component</th> <th>Volume (μL)</th> </tr>
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− | <tr> <td>2x reaction buffer</td> <td>10</td> </tr>
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− | <tr> <td>Construct (ribozyme)</td> <td>1</td> </tr>
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− | <tr> <td>MilliQ H2O</td> <td>6</td> </tr>
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− | <tr> <td>DNA blunting enzyme</td> <td>1</td> </tr>
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− | <tr> <td>pJET vector</td> <td>1</td> </tr>
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− | <tr> <td>T4 DNA ligase</td> <td>1</td> </tr>
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− | </table>
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− | <p>Total volume: 18μL</p>
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− | <ul>
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− | <li>Incubate the mix of the first 4 components at 20°C for 5 minutes</li>
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− | <li>After adding the pJET vector and T4 DNA ligase incubate at room temperature for 5 minutes</li>
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− | </ul>
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− | <h2>Cleaving pSBIC3 </h2>
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− | <table>
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− | <tr> <th>Component</th> <th>Volume (μL)</th> </tr>
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− | <tr> <td>pSBIC3</td> <td>2</td> </tr>
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− | <tr> <td>EcoRI</td> <td>1</td> </tr>
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− | <tr> <td>PstI</td> <td>1</td> </tr>
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− | <tr> <td>Cutsmart buffer 10x</td> <td>2</td> </tr>
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− | <tr> <td>MilliQ H2O</td> <td>14</td> </tr>
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− | </table>
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− | <p>Incubate for 1 hour at 47°C </p>
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− | <h2>Transformations of 1μL construct in pJET1.2 (Devices I, II, III) into 25μL of E. coli DH5α Competent Cells</h2>
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− | <p>Protocol:</p>
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− | <ul>
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− | <li>Add 1μL of DNA to 25μL of competent cells</li>
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− | <li>Incubate for 30 minutes on ice</li>
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− | <li>Heat shock at 47°C for 45 seconds</li>
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− | <li>Incubate on ice for 5 minutes</li>
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− | <li>Add 400μL of LB</li>
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− | <li>Incubate in RNA common room at 37°C for an hour</li>
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− | <li>Plate 400μL on LB + Amp</li>
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− | </ul>
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− | </div>
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− | <div class="entry_expand_button">
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− | More ↓
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− | </div>
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− | </div>
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− | </div>
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− | <!-- End Notebook Entry -->
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− | <!-- Notebook Entry -->
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− | <div class="notebook_entry">
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− | <div class="entry_day">
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− | <div>12</div>
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− | </div>
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− | <div class="entry_content">
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− | <div class="entry_title">
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− | <h1>PCR</h1>
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− | </div>
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− | <div class="entry_data">
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− | <p>Add overnight cultures to LB media with 5μL of Cu antibiotic. Device I colony I (DICI), DIC2, DIIC1, DIICII, DIIICI, DIIICII.</p>
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− | <p>Cultivate overnight cultures to LB media. Test construct in pJET, colonies 1 and 4. Add 5μL of Amp.</p>
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− | <h2>Ligate High and Low Affinity Ribozyme into pJET</h2>
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− | <table>
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− | <tr> <th>Component</th> <th>Volume (μL)</th> </tr>
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− | <tr> <td>2x reaction buffer</td> <td>10</td> </tr>
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− | <tr> <td>Construct</td> <td>1</td> </tr>
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− | <tr> <td>DNA blunting enzyme</td> <td>1</td> </tr>
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− | <tr> <td>MilliQ H2O</td> <td>6</td> </tr>
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− | </table>
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− | <ul>
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− | <li>Ran at 70°C for 5 minutes on a heat block</li>
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− | <li>Add 1μL of pJET blunt vector</li>
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− | <li>Add 1μL of T4 ligase</li>
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− | </ul>
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− | <ol>
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− | <li>1μL of high and low affinity ribozyme added to DH5α cells</li>
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− | <li>Sit on ice for 30 minutes</li>
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− | <li>Heat shock for 45 seconds at 42°C</li>
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− | <li>Put on ice for 5 minutes</li>
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− | <li>Add 400μL of outgrowth media</li>
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− | <li>Incubate for 1 hour in shaker</li>
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− | <li>Plate 200μL on LB + Amp plates</li>
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− | </ol>
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− | <p>Colony PCR of theophylline ribozyme test I using Pfu polymerase</p>
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− | <table>
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− | <tr> <th>Component</th> <th>Volume (μL)</th> <th># of reactions</th> <th>Master Mix</th> </tr>
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− | <tr> <td>MilliQ H2O</td> <td>39</td> <td>4</td> <td>156</td> </tr>
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− | <tr> <td>10X Pfu buffer</td> <td>5</td> <td>4</td> <td>20</td> </tr>
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− | <tr> <td>10 nM dNTPs</td> <td>2</td> <td>4</td> <td>8</td> </tr>
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− | <tr> <td>Pfu polymerase</td> <td>1</td> <td>4</td> <td>4</td> </tr>
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− | <tr> <td>BB prefix primer</td> <td>1</td> <td>4</td> <td>4</td> </tr>
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− | <tr> <td>BB suffix primer</td> <td>1</td> <td>4</td> <td>4</td> </tr>
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− | </table>
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− | <p>1μL of colony in 20μL of dH2O added to each reaction. 1% agarose gel ran using the PCR products</p>
| |
− | </div>
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− | <div class="entry_expand_button">
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− | More ↓
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− | </div>
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− | </div>
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− | </div>
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− | <!-- End Notebook Entry -->
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− |
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− | <!-- Notebook Entry -->
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− | <div class="notebook_entry">
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− | <div class="entry_day">
| |
− | <div>13</div>
| |
− | </div>
| |
− | <div class="entry_content">
| |
− | <div class="entry_title">
| |
− | <h1>Miniprep</h1>
| |
− | </div>
| |
− | <div class="entry_data">
| |
− | <h2>Overnight Cultures Placed in LB Media</h2>
| |
− | <ol>
| |
− | <li>Add 5&L of Amp</li>
| |
− | <li>Low affinity culture 1 (low 1), low 2, low 3, high 1, high 2, high 3</li>
| |
− | <li>Incubate in the shaker</li>
| |
− | </ol>
| |
− | <p>Overnight media cultures test construct 1 and 4, DICI, DICII, DIICI, DIICII, DIIICI, DIIICII were taken off shaker at 10:30am.</p>
| |
− | <p>Miniprep of Ttr 1 and 4, DICI, DICII, DIICI, DIICII, DIIICI and DIIICII.</p>
| |
− | <ol>
| |
− | <li>Add xx mL of culture and centrifuge at xxx</li>
| |
− | <li>Add 100μL of solution I</li>
| |
− | <li>Add 200μL of solution II</li>
| |
− | <li>Add 300μL of solution III</li>
| |
− | <li>Centrifuge</li>
| |
− | <li>Transfer to EE columns, centrifuge at 10,000 rpm for 2 minutes. Discard flow through</li>
| |
− | <li>Add 700μL and centrifuge at 10,000 rpm. Discard flow through was. Repeat once</li>
| |
− | <li>Centrifuge at 10,000 rpm for 1 minute. Discard flow through.</li>
| |
− | <li>Transfer column to 1.5mL tube, add 50μL of MilliQ H2O, centrifuge for 2 min at 10,000 rpm</li>
| |
− | </ol>
| |
− | </div>
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− | <div class="entry_expand_button">
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− | More ↓
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− | </div>
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− | </div>
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− | </div>
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− |
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− | <!-- Notebook Entry -->
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− | <div class="notebook_entry">
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− | <div class="entry_day">
| |
− | <div>17</div>
| |
− | </div>
| |
− | <div class="entry_content">
| |
− | <div class="entry_title">
| |
− | <h1>In Vitro Transcription</h1>
| |
− | </div>
| |
− | <div class="entry_data">
| |
− | <p>In vitro transcription of PCR products of TT ribozyme. As on page 20 using PCRs 1 and 4 as template</p>
| |
− | <ul>
| |
− | <li>Digest of RAP constructs and insertion into pSBIC3 → E+P</li>
| |
− | <li>Digest of TtR test 1 and 4 for insertion into pSBIC3 as well → E+P</li>
| |
− | </ul>
| |
− | <tr> <th>Component</th> <th>Volume (μL)</th> <tr>
| |
− | <tr> <td>pJET plasmid</td> <td>2</td> <tr>
| |
− | <tr> <td>EcoRI</td> <td>1</td> <tr>
| |
− | <tr> <td>PstI</td> <td>1</td> <tr>
| |
− | <tr> <td>Custmart buffer 10x</td> <td>2</td> <tr>
| |
− | <tr> <td>MilliQ H2O</td> <td>14</td> <tr>
| |
− | <p>Total volume: 20μL</p>
| |
− | <h2>Labels</h2>
| |
− | <ol>
| |
− | <li>Tt Test col 1</li>
| |
− | <li>Tt test col 4</li>
| |
− | <li>RAP high 1</li>
| |
− | <li>RAP high 2</li>
| |
− | <li>RAP high 3</li>
| |
− | <li>RAP low 1</li>
| |
− | <li>RAP low 2</li>
| |
− | <li>RAP low 3</li>
| |
− | </ol>
| |
− | <p>In vitro transcriptions in 300μL left overnight at 37°C</p>
| |
− | </div>
| |
− | <div class="entry_expand_button">
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− | More ↓
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− | </div>
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− | </div>
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− | </div>
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− |
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− | <!-- Notebook Entry -->
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− | <div class="notebook_entry">
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− | <div class="entry_day">
| |
− | <div>19</div>
| |
− | </div>
| |
− | <div class="entry_content">
| |
− | <div class="entry_title">
| |
− | <h1>PCR</h1>
| |
− | </div>
| |
− | <div class="entry_data">
| |
− | <p>Streak device I, II and III on CM plates</p>
| |
− | <table>
| |
− | <tr> <th>Component</th> <th>Volume (μL)</th> <tr>
| |
− | <tr> <td>Digest</td> <td>6</td> <tr>
| |
− | <tr> <td>pSBIC3 digest</td> <td>1</td> <tr>
| |
− | <tr> <td>Ligase</td> <td>0.5</td> <tr>
| |
− | <tr> <td>Ligase buffer</td> <td>1</td> <tr>
| |
− | <tr> <td>MilliQ H2O</td> <td>1.5</td> <tr>
| |
− | </table>
| |
− | <p>Total volume: 10μL</p>
| |
− | <p>Put on heat block at 16°C</p>
| |
− | <p>R0040 - Kit plate 2, well 6F</p>
| |
− | <p>I20270 - kit plate 3, well 8P</p>
| |
− | <ul>
| |
− | <li>Resuspend in 10μL of dH2O.</li>
| |
− | <li>Transformed 1μL of DNA to 25μL of DH5α E. coli competent cells.</li>
| |
− | </ul>
| |
− | <p>Protocol</p>
| |
− | <ul>
| |
− | <li>Add 1μL of R0040 to 25μL of competent cells, repeat with I20270</li>
| |
− | <li>Incubate them for 30 minutes on ice</li>
| |
− | <li>Heat shock them at 47°C for 45 seconds</li>
| |
− | <li>Incubate on ice for 5 minutes</li>
| |
− | <li>Add 400μL of LB</li>
| |
− | <li>Incubate in RNA common room at 37°C for an hour</li>
| |
− | <li>Plate 400μL on LB + CM</li>
| |
− | </ul>
| |
− | <p>Cultivate overnight cultures in LB media, 3 colonies for I20270 and R0040 each</p>
| |
− | <ul>
| |
− | <li>Add 5μL of CM</li>
| |
− | <li>Incubate in shaker</li>
| |
− | </ul>
| |
− | <p>Cultivate overnight streak devices in LB media</p>
| |
− | </div>
| |
− | <div class="entry_expand_button">
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− | More ↓
| |
− | </div>
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− | </div>
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− | </div>
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− |
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− | <!-- Notebook Entry -->
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− | <div class="notebook_entry">
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− | <div class="entry_day">
| |
− | <div>20</div>
| |
− | </div>
| |
− | <div class="entry_content">
| |
− | <div class="entry_title">
| |
− | <h1>PCR & Thermo Cycler</h1>
| |
− | </div>
| |
− | <div class="entry_data">
| |
− | <h2>PCR</h2>
| |
− | <table>
| |
− | <tr> <td>Component</tr> <tr>Volume (μL)</td> </tr>
| |
− | <tr> <td>MilliQ dH2O</tr> <tr>39</td> </tr>
| |
− | <tr> <td>10X Pfu buffer</tr> <tr>5</td> </tr>
| |
− | <tr> <td>10 nM dNTPs</tr> <tr>2</td> </tr>
| |
− | <tr> <td>BB prefix/suffix primers</tr> <tr>1</td> </tr>
| |
− | <tr> <td>Template DNA</tr> <tr>0.5</td> </tr>
| |
− | <tr> <td>Pfy pol (Thermo Sci)</tr> <tr>1.0</td> </tr>
| |
− | </table>
| |
− | <h2>Thermo Cycler</h2>
| |
− | <table>
| |
− | <tr> <th>Temperature (°C)</th> <th>Time (seconds)</th> </tr>
| |
− | <tr> <td>98</td> <td>180</td> </tr>
| |
− | <tr> <td>98</td> <td>30</td> </tr>
| |
− | <tr> <td>53</td> <td>30</td> </tr>
| |
− | <tr> <td>72</td> <td>30</td> </tr>
| |
− | <tr> <td>72</td> <td>600</td> </tr>
| |
− | </table>
| |
− | <p>Note: All 30 seconds runs were repeated 35 times.</p>
| |
− | <p>Red → TT test 1</p>
| |
− | <p>Green → TT test 2</p>
| |
− | </div>
| |
− | <div class="entry_expand_button">
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− | More ↓
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− | </div>
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− | </div>
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− | </div>
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− | <!-- End Notebook Entry -->
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− |
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− | <!-- Notebook Entry -->
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− | <div class="notebook_entry">
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− | <div class="entry_day">
| |
− | <div>21</div>
| |
− | </div>
| |
− | <div class="entry_content">
| |
− | <div class="entry_title">
| |
− | <h1>Overexpression</h1>
| |
− | </div>
| |
− | <div class="entry_data">
| |
− | <h2>Overexpression of Devices 1, 2 and 3; and Positive and Negative Controls all with n=6</h2>
| |
− | <table>
| |
− | <tr> <th>Device</th> <th>Sample</th> <th>OD reading for a 1/2 dilution</th> <th>Volume(μL)</th> </tr>
| |
− | <tr> <td rowspan="6">1</td> <td>1</td> <td>0.34</td> <td>0.89</td> </tr>
| |
− | <tr> <td>2</td> <td>0.40</td> <td>0.75</td> </tr>
| |
− | <tr> <td>3</td> <td>0.38</td> <td>0.78</td> </tr>
| |
− | <tr> <td>4</td> <td>0.39</td> <td>0.77</td> </tr>
| |
− | <tr> <td>5</td> <td>0.41</td> <td>0.73</td> </tr>
| |
− | <tr> <td>6</td> <td>0.38</td> <td>0.78</td> </tr>
| |
− | <tr> <td rowspan="6">2</td> <td>1</td> <td>0.38</td> <td>0.78</td> </tr>
| |
− | <tr> <td>2</td> <td>0.39</td> <td>0.77</td> </tr>
| |
− | <tr> <td>3</td> <td>0.43</td> <td>0.70</td> </tr>
| |
− | <tr> <td>4</td> <td>0.40</td> <td>0.75</td> </tr>
| |
− | <tr> <td>5</td> <td>0.33</td> <td>0.91</td> </tr>
| |
− | <tr> <td>6</td> <td>0.38</td> <td>0.78</td> </tr>
| |
− | <tr> <td rowspan="6">3</td> <td>1</td> <td>0.47</td> <td>0.64</td> </tr>
| |
− | <tr> <td>2</td> <td>0.41</td> <td>0.73</td> </tr>
| |
− | <tr> <td>3</td> <td>0.29</td> <td>1.03</td> </tr>
| |
− | <tr> <td>4</td> <td>0.44</td> <td>0.68</td> </tr>
| |
− | <tr> <td>5</td> <td>0.42</td> <td>0.71</td> </tr>
| |
− | <tr> <td>6</td> <td>0.48</td> <td>0.63</td> </tr>
| |
− | <tr> <td rowspan="6">Positive control</td> <td>1</td> <td>0.45</td> <td>0.66</td> </tr>
| |
− | <tr> <td>2</td> <td>0.46</td> <td>0.65</td> </tr>
| |
− | <tr> <td>3</td> <td>0.48</td> <td>0.63</td> </tr>
| |
− | <tr> <td>4</td> <td>0.28</td> <td>1.07</td> </tr>
| |
− | <tr> <td>5</td> <td>0.41</td> <td>0.73</td> </tr>
| |
− | <tr> <td>6</td> <td>0.46</td> <td>0.65</td> </tr>
| |
− | <tr> <td rowspan="6">Negative control</td> <td>1</td> <td>0.41</td> <td>0.73</td> </tr>
| |
− | <tr> <td>2</td> <td>0.46</td> <td>0.65</td> </tr>
| |
− | <tr> <td>3</td> <td>0.44</td> <td>0.68</td> </tr>
| |
− | <tr> <td>4</td> <td>0.41</td> <td>0.73</td> </tr>
| |
− | <tr> <td>5</td> <td>0.44</td> <td>0.68</td> </tr>
| |
− | <tr> <td>6</td> <td>0.42</td> <td>0.71</td> </tr>
| |
− | </table>
| |
− | <ol>
| |
− | <li>3.4/5.6 = 0.6 OD (5μL/5.6) add 0.89μL</li>
| |
− | <li>4/0.6 = 6.66/J add 0.75</li>
| |
− | </ol>
| |
− | </div>
| |
− | <div class="entry_expand_button">
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− | More ↓
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− | </div>
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− | </div>
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− | </div>
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− | <!-- End Notebook Entry -->
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− |
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− | <!-- Notebook Entry -->
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− | <div class="notebook_entry">
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− | <div class="entry_day">
| |
− | <div>24</div>
| |
− | </div>
| |
− | <div class="entry_content">
| |
− | <div class="entry_title">
| |
− | <h1>E. coli Cell Extract</h1>
| |
− | </div>
| |
− | <div class="entry_data">
| |
− | <h2>Preparation of E. coli Cell Extract by Lysozyme</h2>
| |
− | <p>Buffers and solutions: binding/opening buffer DNAse lysozyme sodium deoxycholat</p>
| |
− | <p>Method</p>
| |
− | <ol>
| |
− | <li>Resuspend the cell pellet in 3.5mL of binding/opening buffer by stirring the mixture slowly on ice.</li>
| |
− | <li>Add 350μL of lysozyme and incubate the cell suspension on ice for 30 minutes</li>
| |
− | <li>Add sodium deoxycholat (12.5mg/g)</li>
| |
− | <li>Stir the mixture slowly on ice</li>
| |
− | <li>Add few crystals of DNAse while stirring</li>
| |
− | <li>Remove debris by centrifugation at 3000g for 30 minutes at 4°C</li>
| |
− | <li>Collect the supernatant (cell lysate) in fresh tube</li>
| |
− | <li>Centrifugation for 45 minutes at 30,000g will result in the S-30 extract</li>
| |
− | </ol>
| |
− | <ul>
| |
− | <li>0.5g of cells per tube</li>
| |
− | <li>Lysozyme stock solution</li>
| |
− | <li>20mg/mL lysozyme</li>
| |
− | <li>0.05mL lysozyme solution per mL cell suspension</li>
| |
− | <li>900μL binding buffer</li>
| |
− | <li>12.5mg/g with 0.5g x 18 = 112.5mg</li>
| |
− | </ul>
| |
− | </div>
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− | <div class="entry_expand_button">
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− | More ↓
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− | </div>
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− | </div>
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− | </div>
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− | <!-- End Notebook Entry -->
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− |
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− | <!-- Notebook Entry -->
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− | <div class="notebook_entry">
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− | <div class="entry_day">
| |
− | <div>26</div>
| |
− | </div>
| |
− | <div class="entry_content">
| |
− | <div class="entry_title">
| |
− | <h1>Miniprep</h1>
| |
− | </div>
| |
− | <div class="entry_data">
| |
− | <h2>Miniprep of Tt test 2-1, Tt test 2-2, Tt test 1-1 RAP low 5-3, RAP low 5-1, RAP high 4-1, RAP low 6-1, RAP low 8-1</h2>
| |
− | <ol>
| |
− | <li>Add 100μL of solution I</li>
| |
− | <li>Add 200μL of solution II</li>
| |
− | <li>Add 350μL of solution III</li>
| |
− | <li>Centrifuge at 12,000 rpm for 5 minutes</li>
| |
− | <li>Discard flow through, add 750μL wash solution</li>
| |
− | <li>Centrifuge at 10,000 rpm for 2 minutes. Discard flow through</li>
| |
− | <li>Repeat previous 2 steps</li>
| |
− | <li>Centrifuge at 10,000 rpm for 2 minutes, discard flow through</li>
| |
− | <li>Centrifuge at 10,000 rpm for 1 minute. Discard flow through.</li>
| |
− | <li>Add 50μL of elution buffer (MilliQ H2O), centrifuge at 10,000 rpm for 2 minutes</li>
| |
− | <li>Store DNA at -20°C</li>
| |
− | </ol>
| |
− | <p>Pfu PCR of RAP and Tt test constructs</p>
| |
− | <table>
| |
− | <tr> <th>Component</th> <th>Volume (μL)</th> </tr>
| |
− | <tr> <td>10x Pfu buffer</td> <td>5</td> </tr>
| |
− | <tr> <td>pSBIC3 plasmid + insert</td> <td>p</td> </tr>
| |
− | <tr> <td>10mM dNTPs</td> <td>2</td> </tr>
| |
− | <tr> <td>Forward primer → BB prefix</td> <td>1</td> </tr>
| |
− | <tr> <td>Reverse primer → BB suffix</td> <td>1</td> </tr>
| |
− | <tr> <td>MilliQ dH2O</td> <td>35</td> </tr>
| |
− | <tr> <td>Pfu DNA polymerase</td> <td>1</td> </tr>
| |
− | </table>
| |
− | <p>Total volume: 50μL</p>
| |
− | <table>
| |
− | <tr> <th>Temperature (°C)</th> <th>Time (seconds)</th> </tr>
| |
− | <tr> <td>98</td> <td>180</td> </tr>
| |
− | <tr> <td>98</td> <td>30</td> </tr>
| |
− | <tr> <td>53</td> <td>40</td> </tr>
| |
− | <tr> <td>72</td> <td>40</td> </tr>
| |
− | <tr> <td>72</td> <td>600</td> </tr>
| |
− | </table>
| |
− | <p>Note: All 30 seconds runs were repeated 30 times.</p>
| |
− | <h2>Agarose Gel</h2>
| |
− | <table>
| |
− | <tr> <th>Lane</th> <th>Sample</th> <th>Volume (μL)</th> </tr>
| |
− | <tr> <td>1</td> <td>1 kb ladder</td> <td>3</td> </tr>
| |
− | <tr> <td>2</td> <td>TT test 2-1</td> <td rowspan="8">5μL sample + 1μL 6x dye</td> </tr>
| |
− | <tr> <td>3</td> <td>RAP low RBS 5-3</td> </tr>
| |
− | <tr> <td>4</td> <td>TT test 1-1</td> </tr>
| |
− | <tr> <td>5</td> <td>RAP low RBS 5-1</td> </tr>
| |
− | <tr> <td>6</td> <td>RAP high RBS 4-1</td> </tr>
| |
− | <tr> <td>7</td> <td>TT test 2-2</td> </tr>
| |
− | <tr> <td>8</td> <td>RAP low RBS 8-2</td> </tr>
| |
− | <tr> <td>9</td> <td>RAP low RBS 8-1</td> </tr>
| |
− | <tr> <td>10</td> <td>1 kb ladder</td> <td>3</td> </tr>
| |
− | </table>
| |
− | </div>
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− | More ↓
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