Team:Duke/Notebook

From 2014.igem.org

(Difference between revisions)
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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>
 
-
<li>Expected band size 715 bp </li>
 
-
<li>Control (no template) showed no band, lane 4 produced faint band </li>
 
-
<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>
 
-
<div class="lab">
 
-
 
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<p> PCR of Z4EV from pMN10</p>
 
-
<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>
 
-
<div class="lab">
 
-
 
-
<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>
 
-
<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>
 
-
<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>
 
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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>
 
<div class="day">
<div class="day">

Revision as of 15:58, 29 July 2014


Lab Notebook

April 2014
Month 1 of Project
sun mon tue wed thu fri sat
1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30
May 2014
Month 2 of Project
sun mon tue wed thu fri sat
1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
June 2014
Month 3 of Project
sun mon tue wed thu fri sat
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30
July 2014
Month 4 of Project
sun mon tue wed thu fri sat
1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August 2014
Month 5 of Project
sun mon tue wed thu fri sat
1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September 2014
Month 6 of Project
sun mon tue wed thu fri sat
1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October 2014
Month 7 of Project
sun mon tue wed thu fri sat
1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31

April Overview
Objectives

This is what we were trying to accomplish in April.

May, June Overview
Objectives

This is what we were trying to accomplish in May and June.

May 29
Objective: Prepare Chemically competent E. Coli
Matthew Faw

Followed Charlie’s Cloning Protocols to prepare chemically competent E. Coli

  • 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

Next Steps:

  • Transformation

June 1
Objective: Transform Chemically competent E. Coli (CCEC)
Matthew Faw

Transformation of CCEC with RFP Construct of varying concentrations+control with no DNA

  • Followed Charlie’s Cloning Protocols
    • Mistakenly used all of DNA construct from the kit… So not sure if the transformation will be successful
  • Plated the transformed DNA and put in incubator at 37C
  • Results (6/2/14)--Transformation unsuccessful because improper procedure was done by MFaw

Next Steps:

  • Look at plates, compare cultures

June 2
Objective: Redo yesterday’s transformation
Matthew Faw

Due to my failure to correctly conduct the transformation yesterday, it was necessary to redo the transformation.

  • Followed Charlie’s Cloning Protocols
    • Added 1 ul of .5, 5, 10, 20, 50, and 0 (control) pg/lof RFP Construct to chemically competent E. Coli (CCEC), and followed procedure
  • Plated the transformed DNA on 6 separate plates, put in 37C overnight
  • Results: (6/3/14)-Transformation unsuccessful. Try the transformation again with Charlie’s cells and with new cells grown using iGEM’s protocol

Next Steps:

  • Look at plates, compare cultures, etc.
6/4/14 Objective: Prepare buffers and mediums for new CCEC protocol Matthew Farnitano, TJ Ciesla, Mike Zhu, Charlie Cooper Prepare SOB Medium for bacterial transformation Protocol from iGEM’s parts website http://parts.igem.org/Help:Protocols/Competent_Cells Made 1 L, autoclaved and stored in two 500 mL containers in cold room medium still appeared cloudy before autoclave--may just be new recipe Prepare CCMB 80 Buffer for making chemically competent E. coli cells Protocol from iGEM’s parts website http://parts.igem.org/Help:Protocols/Competent_Cells Made 1 L, filtered and stored in two 500 mL containers in cold room pH 6.34 (overshot a few times pH adjustment, but no noticeable precipitate formed Autoclave two 500 mL culture flasks For CCEC protocol With water inside to remove detergent residues Next steps: Prepare CCEC Objective: Attempt to grow some transformed cell cultures with Charlie’s CCEC Matthew Faw The second (and more properly conducted) attempt to transform the CCEC with RFP construct BBa_J04450 from 6/2 failed. Today, we are trying to see if we can get any transformed cells to grow in plates. Followed Charlie’s Cloning protocols,with slight modifications Added 5 lof 0 (control) and 50pg/lRFP Construct BBa_J04450 to Charlie’s chemically competent E. Coli, and followed procedure Plated the transformed DNA on 2 separate plates, put in put in 37C overnight Results: No colonies grew (6/5/14) Next Steps: -Examine plates to see if any cultures grew -Attempt the transformation with the cell cultures growns using iGEM’s standard protocols that can be found here: http://parts.igem.org/Help:Protocols/Competent_Cells -Lab currently in the process of making these CCEC Objective: Prepare pdCas9 marrafini and pCsy4 Arkin plasmids to be miniprepped Matthew Faw, Charlie Cooper Prepare pdCas9 and pCsy4 to be miniprepped tomorrow Put pdCas9 into a culture tube with 5ml SOC+Chloramphenicol Put pCsy4 in a culture tube with 5ml SOC+Amp Left both to shake at 37C to grow the plasmid DNA--Charlie will retrieve them Next steps: Miniprep plasmid DNA

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