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Team:Duke/Notebook
From 2014.igem.org
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<div class="obj">Objective: Create Z4EV-dCas9-Mxi1 construct</div> | <div class="obj">Objective: Create Z4EV-dCas9-Mxi1 construct</div> | ||
<div class="ppl">Matthew Farnitano and Matthew Faw</div> | <div class="ppl">Matthew Farnitano and Matthew Faw</div> | ||
| - | + | <div class="lab> | |
| - | < | + | <ul> |
| + | <li>PCR of Z4EV from pMN10</li> | ||
<ul> | <ul> | ||
<li>new oligos SpeI-Z4EV5p and NcoI-spc-Z4EV3p (diluted to 100uM) </li> | <li>new oligos SpeI-Z4EV5p and NcoI-spc-Z4EV3p (diluted to 100uM) </li> | ||
| Line 32: | Line 33: | ||
</ul> | </ul> | ||
| - | < | + | <li>Results (04/08/2014): Lanes 2 and 3 produced band at ~0.7-0.8 kb (agarose gel)</li> |
<ul> | <ul> | ||
<li>Expected band size 715 bp </li> | <li>Expected band size 715 bp </li> | ||
| Line 39: | Line 40: | ||
</ul> | </ul> | ||
| - | < | + | <li>Next steps: </li> |
<ul> | <ul> | ||
<li>Run on gel to confirm (4/8) </li> | <li>Run on gel to confirm (4/8) </li> | ||
<li>digest PCR product and dCas9/Mxi1 with SpeI/NcoI to ligate (4/8) </li> | <li>digest PCR product and dCas9/Mxi1 with SpeI/NcoI to ligate (4/8) </li> | ||
</ul> | </ul> | ||
| - | + | </ul> | |
</div> | </div> | ||
Revision as of 17:14, 28 July 2014
April 7Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano and Matthew Faw
April 8Objective: Create Z4EV-dCas9-Mxi1 construct Matthew Farnitano Agarose Gel with Z4EV PCR products PCR from 04/07/2014 of Z4EV from pMN10 Results (04/08/2014): Lanes 2 and 3 produced band at ~0.7-0.8 kb (agarose gel) Expected band size 715 bp Control (no template) showed no band, lane 4 produced faint band Lanes 3 and 4 showed strong band at ~5-6 kb (template expected 5.1 kb) PCR cleanup of Z4EV PCR products PCR from 04/07/2014 of Z4EV from pMN10 Used only lanes 2 and 3 (successful from gel) Concentration 32.4 ng/uL in 50 uL Restriction Digest of Z4EV PCR product and TDH3-dCas9-Mxi1 Both with SpeI-HF and NcoI-HF in Cutsmart PCR from 04/07/2014 of Z4EV from pMN10 50 uL (entire product) in 65 uL reaction dCas9 plasmid construct from CC 190ng/uL 20 uL in 65 uL reaction PCR cleanup of Z4EV PCR digest product 04/08 digest of 04/07 PCR Results: Final concentration negligible, no DNA Next steps: Z4EV PCR again from pMN10 Digest of PCR in SpeI/NcoI Gel extraction of digested TDH3-dCas9-MxiI April 10 Aliquam vestibulum ornare mauris, tincidunt rhoncus orci euismod a. Maecenas mollis felis et leo varius, at blandit turpis auctor. Nam at dictum quam, sit amet vehicula ligula. Nullam faucibus neque ac libero tempor malesuada. Quisque semper turpis luctus odio condimentum rutrum. Cras sodales ultrices condimentum. Aliquam ultrices risus at diam pretium, quis bibendum lacus elementum. Duis tincidunt nunc nunc, vitae sodales orci luctus eget. Proin ante enim, facilisis vel sapien nec, dapibus suscipit sapien. Mauris aliquet sodales lobortis. In tempus neque magna, consectetur mollis turpis laoreet dignissim. Vivamus a dui turpis. Nullam libero arcu, vestibulum sed tellus ac, ornare semper elit. Quisque facilisis egestas cursus. Curabitur fermentum leo ut odio dignissim euismod. Integer pellentesque vestibulum neque, sit amet malesuada nunc sollicitudin vel. Curabitur a dui cursus libero cursus sodales nec lobortis leo. Nulla tempor volutpat eros, id tristique felis pellentesque nec. Etiam ultrices ligula ut eros viverra, eu pharetra lacus volutpat. Aliquam mattis lobortis leo, sed ornare lacus. Mauris vel gravida velit. Quisque ut viverra nisl, eget consectetur enim. In commodo imperdiet euismod. In rhoncus a enim ut sollicitudin. Praesent mattis dolor a erat euismod interdum. In a neque non enim sollicitudin fringilla quis sit amet justo. Nam mauris massa, tristique sit amet semper sit amet, pharetra eget felis. Sed in tortor purus. Donec elit nunc, commodo vel mollis in, iaculis id nunc. Praesent ut convallis dui, a bibendum dui. Donec non malesuada quam. Duis sem urna, tincidunt sit amet odio sed, gravida tincidunt enim. Cras risus elit, laoreet vitae feugiat non, viverra vel eros. Suspendisse potenti. Maecenas vel suscipit eros. |
