Team:Duke/Notebook

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<table padding="50px">
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<div class="month"><a href="https://2014.igem.org/Team:Duke/Notebook/Overview">Overview of Each Month </a></div>
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<tr>
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<div class="month"><a href="https://2014.igem.org/Team:Duke/Notebook/Months">Notebook Entries by Month</a></div>
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<td>
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<div class="month"><a href="https://2014.igem.org/Team:Duke/Notebook/Protocols">Protocols</a></div>
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</td>
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<div class="rightnb">
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<div class="day">
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<a id="apr"><h1>April - Overview</h1></a>
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<div class="obj">Objectives</div>
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<div class="lab">
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<p>This is what we were trying to accomplish in April. </p>
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</div>
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<div class="day">
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<a id="apr7"><h2> April 7 </h2></a>
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<div class="obj">Objective: Create Z4EV-dCas9-Mxi1 construct</div>
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<div class="ppl">Matthew Farnitano and Matthew Faw</div>
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<div class="lab">
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<p>PCR of Z4EV from pMN10</p>
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<ul>
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<li>new oligos SpeI-Z4EV5p and NcoI-spc-Z4EV3p (diluted to 100uM) </li>
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<li>4 tubes with 0, 0.3, 0.6, 1 uL template </li>
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<ul><li>iGEM 3-step protocol with 65C anneal, 20 sec extend</li></ul>
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<li>Note: did not dilute template beforehand (do this in future)</li>
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</ul>
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<p>Results (04/08/2014): Lanes 2 and 3 produced band at ~0.7-0.8 kb (agarose gel)</p>
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<ul>
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<li>Expected band size 715 bp </li>
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<li>Control (no template) showed no band, lane 4 produced faint band </li>
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<li>Lanes 3 and 4 showed strong band at ~5-6 kb (template expected 5.1 kb) </li>
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</ul>
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<p>Next steps: </p>
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<ul>
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<li>Run on gel to confirm (4/8) </li>
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<li>digest PCR product and dCas9/Mxi1 with SpeI/NcoI to ligate (4/8) </li>
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</ul>
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</div>
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</div>
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<div class="day">
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<h2><a id="apr8"> April 8 </a></h2>
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<div class="obj">Objective: Create Z4EV-dCas9-Mxi1 construct</div>
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<div class="ppl">Matthew Farnitano</div>
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<div class="lab">
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<p> Agarose Gel with Z4EV PCR products </p>
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<ul>
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<li>PCR from 04/07/2014 of Z4EV from pMN10</li>
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<li>Results (04/08/2014): Lanes 2 and 3 produced band at ~0.7-0.8 kb (agarose gel) </li>
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<ul>
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<li>Expected band size 715 bp </li>
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<li>Control (no template) showed no band, lane 4 produced faint band </li>
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<li>Lanes 3 and 4 showed strong band at ~5-6 kb (template expected 5.1 kb) </li>
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</ul>
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</ul>
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<p>PCR cleanup of Z4EV PCR products</p>
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<ul>
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<li>PCR from 04/07/2014 of Z4EV from pMN10 </li>
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<li>Used only lanes 2 and 3 (successful from gel) </li>
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<li>Concentration 32.4 ng/uL in 50 uL </li>
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</ul>
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<p> Restriction Digest of Z4EV PCR product and TDH3-dCas9-Mxi1</p>
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<ul>
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<li>Both with SpeI-HF and NcoI-HF in Cutsmart </li>
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<li>PCR from 04/07/2014 of Z4EV from pMN10 -- 50 uL (entire product) in 65 uL reaction </li>
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<li>dCas9 plasmid construct from CC 190ng/uL -- 20 uL in 65 uL reaction </li>
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</ul>
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<p> PCR cleanup of Z4EV PCR digest product</p>
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<ul>
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<li>04/08 digest of 04/07 PCR</li>
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<li> Results: Final concentration negligible, no DNA</li>
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</ul>
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<p> Next steps: </p>
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<ul>
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<li>Z4EV PCR again from pMN10</li>
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<li>Digest of PCR in SpeI/NcoI</li>
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<li>Gel extraction of digested TDH3-dCas9-MxiI</li>
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</ul>
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</div>
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</div>
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<div class="day">
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<a id="apr10"><h2> April 10 </h2></a>
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<div class="obj">Objective: Create Z4EV-dCas9-Mxi1 construct</div>
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<div class="ppl">Matthew Farnitano</div>
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<div class="lab">
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<p> PCR of Z4EV from pMN10</p>
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<ul>
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<li>Repeat of PCR from 4/7/14</li>
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<li>Oligos SpeI-ZeEV5p and NcoI-Spc-Z4EV3p</li>
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<li>Diluted pMN10 template to 1 ng/uL before use</li>
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<li>8 tubes: 0, 0.3, 0.5, 0.5, 0.7, 0.7, 1.0, 1.0 uL template in 50 uL reaction</li>
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<ul><li>iGEM 3-step protocol with 65C anneal, 20 sec extend</li></ul>
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<li>Results (see 4/11): expected bands present, but concentrations too low</li>
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</ul>
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<p> Gel Extraction of backbone (-TDH3) from TDH3-dCas9-Mxi1 digest</p>
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<ul>
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<li>Digest performed 4/8 with SpeI/NcoI</li>
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<li>Expected band sizes: 10.5 kb (backbone, desired piece), 637 bp (TDH3, removed piece)</li>
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<li>Obtained consistent band sizes, used 400 mg gel in extraction <</li>
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<li>Gel picture: <img src="https://static.igem.org/mediawiki/2014/3/3c/4-10-14gel.png"></li>
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<li> Results: obtained 20 uL product at 55 ng/uL (froze for later use)</li>
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</ul>
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<!-- https://2014.igem.org/File:4-10-14gel.png or https://static.igem.org/mediawiki/2014/3/3c/4-10-14gel.png -->
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<p> Next steps: </p>
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<ul>
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<li> Run gel of PCR</li>
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<li> Clean up PCR</li>
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</ul>
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</div>
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</div>
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<div class="day">
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<a id="apr11"><h2>April 11</h2></a>
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<div class="obj">Objective: Create Z4EV-dCas9-Mxi1 construct</div>
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<div class="ppl"> Matthew Farnitano and Matthew Faw</div>
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<div class="lab">
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<p> Agarose gel to analyze PCR of Z4EV from pMN10</p>
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<ul>
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<li>PCR from 4/10</li>
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<li>Expected band size: 715 bp</li>
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<li>Showed band at expected size in all seven non-control lanes</li>
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<li>Intensities appear low, increase in higher lanes (higher template conc.)</li>
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<li>Gel picture: <img src="https://static.igem.org/mediawiki/2014/c/cd/4-11-14gel.png"> </li>
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<!-- https://2014.igem.org/File:4-11-14gel.png or https://static.igem.org/mediawiki/2014/c/cd/4-11-14gel.png -->
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</ul>
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<p>PCR cleanup of Z4EV from 4/10 PCR</p>
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<ul>
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<li>final concentration from 7 tubes: 25.8 ng/uL in 30 uL</li>
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<li>Need to improve</li>
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</ul>
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<p> Next Steps:</p>
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<ul>
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<li>Redo PCR of Z4EV. Things to try:</li>
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<ul>
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<li>Use previous cleanup as a template</li>
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<li>use 1 uL enzyme instead of 0.5</li>
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<li>slightly longer extension time</li>
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<li>higher template concentration</li>
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</ul>
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<li>PCR cleanup: Things to try:</li>
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<ul>
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<li>Run PB buffer flow-through back through filter during step 7</li>
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<li>let PE buffer sit in column (covered) while ethanol evaporates</li>
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<li>let elution buffer sit on DNA for 2-3 minutes before spin</li>
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</ul>
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</ul>
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</div>
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</div>
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<div class="day">
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<a id="apr13"><h2>April 13 - Unfinished</h2></a>
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<div class="obj">Objective: Create Z4EV-dCas9-Mxi1 construct</div>
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<div class="ppl"> Matthew Farnitano and Garima Tomar</div>
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<div class="lab">
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<p> PCR of Z4EV from pMN10</p>
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<ul>
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<li>Third attempt (previously 4/7, 4/10)</li>
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<li>oligos SpeI-Z4EV5p and NcoI-spc-Z4EV3p</li>
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<li>8 identical tubes with 1.5 uL template (1 ng/uL stock) in 50 uL reaction</li>
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<ul>
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<li>Used 1 uL polymerase (instead of 0.5 uL) per 50 uL reaction</li>
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<li>iGEM 3-step protocol with 65C anneal, 1 min extension</li>
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</ul>
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<li>Changes from previous: more template, more polymerase, longer extension</li>
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</ul>
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<p> Results (Date): </p>
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<ul>
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<li>???</li>
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</ul>
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<p> Next steps: </p>
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<ul>
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<li>Run on gel</li>
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<li>Clean up PCR with new notes (see 4/11), see if concentration is better </li>
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</ul>
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</div>
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</div>
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<div class="day">
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<a id="apr14"><h2>April 14</h2></a>
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<div class="obj">Objective: Create Z4EV-dCas9-Mxi1 construct</div>
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<div class="ppl">Matthew Farnitano</div>
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<div class="lab">
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<p> Agarose gel of Z4EV PCR</p>
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<ul>
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<li>Z4EV from pMN10, 4/13 PCR</li>
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<li>2-log ladder and 8 identical reaction tubes</li>
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<li>Expected band size ~715 bp</li>
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<ul>
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<li>All lanes showed band 700-800 bp</li>
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<li>band intensity much greater than previous attempts</li>
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</ul>
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<li>Unable to take normal picture: used dark room and iPhone camera to photograph</li>
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</ul>
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<p>PCR cleanup of Z4EV PCR from 4/13</p>
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<ul>
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<li>Qiagen miniprep steps 7-10</li>
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<li>8 tubes into 1 final product</li>
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<li>Modifications to protocol:</li>
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<ul>
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<li>ran PB buffer through twice</li>
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<li>let column sit, covered, for 10-15 min after PE buffer wash/discard step</li>
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<li>let water for elution sit on column for 3 min before spin</li>
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</ul>
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<li>Results: Obtained 30 uL product at 407.5 ng/uL (in gel fridge for immediate use)</li>
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</ul>
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<p>Next Steps: </p>
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<ul>
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<li>Restriction digest of Z4EV and PCR cleanup with SpeI and NcoI</li>
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<li>ligation of Z4EV with dCas9 backbone (obtained 4/10)</li>
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<li>transformation</li>
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</ul>
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</div>
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</div>
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<div class="day">
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<a id="apr15"><h2>April 15</h2></a>
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<div class="obj">Objective: Create Z4EV-dCas9-Mxi1 construct</div>
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<div class="ppl">Matthew Farnitano</div>
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<div class="lab">
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<p> Restriction Digest of Z4EV-PCR with SpeI/NcoI</p>
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<ul>
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<li>PCR from 4/13, cleaned 4/14</li>
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<li>Cut with SpeI-HF and NcoI-HF in Cutsmart buffer</li>
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<ul>
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<li>30 uL PCR product (407.5 ng/uL) in 50 uL reaction</li>
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<li>Incubated at 37C for 4.5 hrs</li>
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</ul>
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</ul>
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<p> PCR Cleanup of Restriction Digest of Z4EV</p>
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<ul>
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<li>Qiagen kit with modified methods described on 4/14 </li>
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<li>Results: Obtained 30 uL product at 248.1 ng/uL DNA (nanodrop) </li>
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</ul>
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<p> Ligation of Z4EV PCR and dCas9-MxiI backbone</p>
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<ul>
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<li>Z4EV from pMN10, digested with NcoI and SpeI (4/15)</li>
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<li>dCas9-MxiI backbone digested with NcoI and SpeI, extracted 4/10</li>
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<li>3:1 molar ratio backbone:insert</li>
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<ul>
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<li>20 uL total reaction - 15.3 uL backbone (55 ng/uL), 0.7 uL insert (248.1 ng/uL)</li>
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</ul>
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<li>Left in cold room overnight in 16C heat block</li>
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</ul>
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<p>Next steps: </p>
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<ul>
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<li>Transform ligated plasmid into E. coli, clone, then miniprep.</li>
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</ul>
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</div>
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<div class="day">
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<a id="apr16"><h2>April 16</h2></a>
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<div class="obj">Objective: Create Z4EV-dCas9-Mxi1 construct</div>
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<div class="ppl">Matthew Farnitano and Charlie Cooper</div>
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<div class="lab">
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<p> Transformation of ligated Z4EV-dCas9-MxiI plasmid into E. coli</p>
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<ul>
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<li>Ligation left overnight for 22 hrs</li>
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<li>Heat shock protocol with chemically competent E. coli</li>
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<ul>
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<li>1 sample plus 1 no-insert control</li>
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<li>grown in SOC medium, plated on LB+Amp plates</li>
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</ul>
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<li> Results (Charlie Cooper) (4/17): No colonies on either plate</li>
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<ul>
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<li>Possibly used ineffective cells</li>
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</ul>
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</ul>
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<p> Next steps: </p>
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<ul>
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<li>Digest and gel extraction of backbone</li>
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<li>Ligation and Transformation</li>
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</ul>
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</div>
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</div>
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<a id="may29"><h2>May 29</h2></a>
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<div class="obj">Objective: Prepare Chemically competent E. Coli</div>
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<div class="ppl">Matthew Faw</div>
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<div class="lab">
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<p> Followed Charlie’s Cloning Protocols to prepare chemically competent E. Coli </p>
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<ul>
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<li> Once E. Coli were prepared, the solution was aliquoted into 12 labeled tubes (450l in each tube) and tubes were stored in iGEM box in -80C cooler on far left</li>
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</ul>
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<p>Next Steps: </p>
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<ul>
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<li> Transformation </li>
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</ul>
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</div>
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Latest revision as of 02:14, 18 October 2014

Overview of Each Month
Notebook Entries by Month
Protocols

Retrieved from "http://2014.igem.org/Team:Duke/Notebook"