Difference between revisions of "Team:EPF Lausanne/Notebook/Ecoli"

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                 <div class="col-md-12 text-center">
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                     <h3>Protocols</h3>
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                     <h3>E. Coli Laboratory Notebook</h3>
 
                 </div>
 
                 </div>
 
             </div>
 
             </div>
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                             </ul>
 
                             </ul>
 
                         </li>
 
                         </li>
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                         <li><a href="#pdcas9wsgrna">Make pdCas9-w-sgRNAs</a></li>
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                         <li class="dropdown">
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                            <a class="dropdown-toggle" data-toggle="dropdown" href="#pdcas9w">Make pdCas9-w<span class="caret"></span></a>
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                                <ul class="dropdown-menu">
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                                    <li><a href="#PCRpdcas9w">PCR</a></li>
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                                    <li><a href="#taqpcr">Taq PCR</a></li>                   
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                                </ul>
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                        </li>
 
                     </ul>
 
                     </ul>
 
                 </nav>
 
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             <div class="col-sm-9">
 
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     <!-- MAKE PDCAS9-W -->
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     <!-- PCR PDCAS9 AND W-->
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                <div id="pdcas9w" class="panel">   
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                    <h1>Make pdCas9-w</h1>
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                        <p>Fuse dCas9 (from plasmid pdCas9) with the w subunit of polymerase (from plasmid pWJ66) to allow dCas9 to activate a gene when it binds upstream of the promoter.</p>
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                        <p>We received the plasmids in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate the plasmids.</p>
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                        <p>We opened pdCas9 and extracted the w subunit from pWJ66 by PCR. We assembled these parts with Gibson assembly.</p>
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                </div>
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−
 
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    <!-- PCR PDCAS9 -->
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                 <div id="PCRpdcas9" class="panel">  
 
                 <div id="PCRpdcas9" class="panel">  
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                     <h2>Open pdCas9 by PCR and extract w subunit from pWJ66 by PCR</h2>
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                     <h1>Make pdCas9-w:</br>Open pdCas9 and extract w subunit from pWJ66 by PCR</h2>
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                         <h3>05.06.2015</h3>
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                         <p><small>We received plasmids pdCas9 and pWJ66 in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate them.</small></p>
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                             <h4>Materials and method</h4>
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                        <h2>05.06.2015</h2>
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                                 <p>Phusion PCR of 1 ng pdCas9 with HF buffer, annealing temperature (Ta): 58°C, extension time (ext): 150 sec, 30 cycles</p>
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                             <h3>Materials and method</h3>
−
                                <p>Phusion PCR of 1 ng pWJ66 with HF buffer, annealing temperature (Ta): 62°C, extension time (ext): 15 sec, 30 cycles</p>
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                                 <ul>
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                                <p>PCR products were analyzed after PCR Product Purification (cf. Protocols) by 1,2% agarose gel electrophoresis.</p>
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                                    <li>20 µl Phusion PCR (cf. Protocols) with 1 ng pdCas9 and 1ng of pWJ66, HF buffer and corresponding primers</li>
−
                            <h4>Results</h4>
+
                                    <li>PCR product purification (cf. Protocols)</li>
 +
                                    <li>Agarose gel electrophoresis of purified PCR products</li>
 +
                                </ul>
 +
                            <h3>Results</h3>
 +
                                <img src="http://https://static.igem.org/mediawiki/2015/e/ee/7.05_pcr_gel.jpg">
 +
                                <p><small>Gel shows that extraction of w subunit from pWJ66 was successful, but opening of pdCas9 was not.</br></small></p>
 +
                        <h2>05.10.2015</h2>
  
 +
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                </div>
  
  

Revision as of 19:14, 27 July 2015

E. Coli Laboratory Notebook

Make pdCas9-w:
Open pdCas9 and extract w subunit from pWJ66 by PCR

We received plasmids pdCas9 and pWJ66 in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate them.

05.06.2015

Materials and method

  • 20 µl Phusion PCR (cf. Protocols) with 1 ng pdCas9 and 1ng of pWJ66, HF buffer and corresponding primers
  • PCR product purification (cf. Protocols)
  • Agarose gel electrophoresis of purified PCR products

Results

Gel shows that extraction of w subunit from pWJ66 was successful, but opening of pdCas9 was not.

05.10.2015

Still under construction

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