Team:Duke/Notebook

From 2014.igem.org

(Difference between revisions)
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<div class="day">
<div class="day">
<h2><a id="apr8"> April 8 </a></h2>
<h2><a id="apr8"> April 8 </a></h2>
-
<p class="obj">Objective: Create Z4EV-dCas9-Mxi1 construct</p>
+
<div class="obj">Objective: Create Z4EV-dCas9-Mxi1 construct</div>
-
Matthew Farnitano
+
<div class="ppl">Matthew Farnitano</div>
-
Agarose Gel with Z4EV PCR products
+
<div class="lab">
-
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)
+
<p> Agarose Gel with Z4EV PCR products </p>
-
Expected band size 715 bp
+
<ul>
-
Control (no template) showed no band, lane 4 produced faint band  
+
<li>PCR from 04/07/2014 of Z4EV from pMN10</li>
-
Lanes 3 and 4 showed strong band at ~5-6 kb (template expected 5.1 kb)
+
<li>Results (04/08/2014): Lanes 2 and 3 produced band at ~0.7-0.8 kb (agarose gel) </li>
-
PCR cleanup of Z4EV PCR products
+
<ul>
-
PCR from 04/07/2014 of Z4EV from pMN10
+
<li>Expected band size 715 bp </li>
-
Used only lanes 2 and 3 (successful from gel)
+
<li>Control (no template) showed no band, lane 4 produced faint band </li>
-
Concentration 32.4 ng/uL in 50 uL
+
<li>Lanes 3 and 4 showed strong band at ~5-6 kb (template expected 5.1 kb) </li>
-
Restriction Digest of Z4EV PCR product and TDH3-dCas9-Mxi1
+
</ul>
-
Both with SpeI-HF and NcoI-HF in Cutsmart
+
</ul>
-
PCR from 04/07/2014 of Z4EV from pMN10
+
 
-
50 uL (entire product) in 65 uL reaction
+
<p>PCR cleanup of Z4EV PCR products</p>
-
dCas9 plasmid construct from CC 190ng/uL
+
<ul>
-
20 uL in 65 uL reaction
+
<li>PCR from 04/07/2014 of Z4EV from pMN10 </li>
-
PCR cleanup of Z4EV PCR digest product
+
<li>Used only lanes 2 and 3 (successful from gel) </li>
-
04/08 digest of 04/07 PCR
+
<li>Concentration 32.4 ng/uL in 50 uL </li>
-
Results: Final concentration negligible, no DNA
+
</ul>
-
Next steps:  
+
 
-
Z4EV PCR again from pMN10
+
<p> Restriction Digest of Z4EV PCR product and TDH3-dCas9-Mxi1</p>
-
Digest of PCR in SpeI/NcoI
+
<ul>
-
Gel extraction of digested TDH3-dCas9-MxiI
+
<li>Both with SpeI-HF and NcoI-HF in Cutsmart </li>
 +
<li>PCR from 04/07/2014 of Z4EV from pMN10 -- 50 uL (entire product) in 65 uL reaction </li>
 +
<li>dCas9 plasmid construct from CC 190ng/uL -- 20 uL in 65 uL reaction </li>
 +
</ul>
 +
 
 +
<p> PCR cleanup of Z4EV PCR digest product</p>
 +
<ul>
 +
<li>04/08 digest of 04/07 PCR</li>
 +
<li> Results: Final concentration negligible, no DNA</li>
 +
</ul>
 +
 
 +
<p> Next steps: </p>
 +
<ul>
 +
<li>Z4EV PCR again from pMN10</li>
 +
<li>Digest of PCR in SpeI/NcoI</li>
 +
<li>Gel extraction of digested TDH3-dCas9-MxiI</li>
 +
</ul>
 +
</div>
 +
</div>
 +
 
 +
 
<a id="apr10"> April 10 </a>
<a id="apr10"> April 10 </a>

Revision as of 17:25, 28 July 2014


April 7

Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano and Matthew Faw

April 8

Objective: 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.

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