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="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"></div>
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<div class="lab">
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<p></p>
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<ul>
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<li></li>
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</ul>
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</div>
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</div>
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04/15/14
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Matthew Farnitano
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Restriction Digest of Z4EV-PCR with SpeI/NcoI
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PCR from 4/13, cleaned 4/14
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Cut with SpeI-HF and NcoI-HF in Cutsmart buffer
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30 uL PCR product (407.5 ng/uL) in 50 uL reaction
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Incubated at 37C for 4.5 hrs
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PCR Cleanup of Restriction Digest of Z4EV
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Qiagen kit with modified methods described on 4/14
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Results: Obtained 30 uL product at 248.1 ng/uL DNA (nanodrop)
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Ligation of Z4EV PCR and dCas9-MxiI backbone
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Z4EV from pMN10, digested with NcoI and SpeI (4/15)
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dCas9-MxiI backbone digested with NcoI and SpeI, extracted 4/10
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3:1 molar ratio backbone:insert
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20 uL total reaction
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15.3 uL backbone (55 ng/uL)
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0.7 uL insert (248.1 ng/uL)
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Left in cold room overnight in 16C heat block
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Next steps:
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Transform ligated plasmid into E. coli, clone, then miniprep.
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04/16/14
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Objective: Create Z4EV-dCas9-Mxi1 construct
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Matthew Farnitano
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Transformation of ligated Z4EV-dCas9-MxiI plasmid into E. coli
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Ligation left overnight for 22 hrs
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Heat shock protocol with chemically competent E. coli
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1 sample plus 1 no-insert control
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grown in SOC medium, plated on LB+Amp plates
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Results (Charlie Cooper) (4/17): No colonies on either plate
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Possibly used ineffective cells
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Next steps:
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Digest and gel extraction of backbone
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Ligation and Transformation
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<div class="day">
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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"