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

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                     <ul class="nav nav-pills nav-stacked" data-spy="affix" data-offset-top="200" data-offset-bottom="600">
 
                     <ul class="nav nav-pills nav-stacked" data-spy="affix" data-offset-top="200" data-offset-bottom="600">
 
                         <li class="dropdown">
 
                         <li class="dropdown">
                             <a class="dropdown-toggle" data-toggle="dropdown" href="#">Make pdCas9-w<span class="caret"></span></a>
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                             <a class="dropdown-toggle" data-toggle="dropdown" href="#">Assemble pdCas9-w<span class="caret"></span></a>
 
                                 <ul class="dropdown-menu">
 
                                 <ul class="dropdown-menu">
                                     <li><a href="#PCRpdcas9">PCR</a></li>
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                                     <li><a href="#PCRpdcas9">Open pdCas9 by PCR</a></li>
                                     <li><a href="#taqpcr">Taq PCR</a></li>                    
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                                     <li><a href="#PCRpwj66">Extract w subunit from pWJ66 by PCR</a></li>
 
                                 </ul>
 
                                 </ul>
 
                         </li>
 
                         </li>
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             <div class="col-sm-9">
 
             <div class="col-sm-9">
  
     <!-- PCR PDCAS9 AND W-->
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     <!-- PCR PDCAS9-->
 
                 <div id="PCRpdcas9" class="panel">  
 
                 <div id="PCRpdcas9" class="panel">  
                     <h1>Make pdCas9-w:</br>Open pdCas9 and extract w subunit from pWJ66 by PCR</h2>
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                     <h1>Assemble pdCas9-w:</br>Open pdCas9 by PCR</h2>
                         <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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                         <p><small>We received plasmid pdCas9 in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate it.</small></p>
                         <h2>05.06.2015</h2>
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                         <h2>Materials and method</h2>
                            <h3>Materials and method</h3>
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                                <ul>
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                                    <li>20 µl Phusion PCR (cf. Protocols) with 1 ng pdCas9/pWJ66, HF buffer, corresponding primers, 30 cycles, annealing temperature 58°C/62°C and extension time 150/15 seconds</li>
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                                    <li>PCR product purification (cf. Protocols)</li>
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                                    <li>Agarose gel electrophoresis of purified PCR products</li>
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                                </ul>
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                            <h3>Results</h3>
+
                                <p align="center"><img src="https://static.igem.org/mediawiki/2015/e/ee/7.05_pcr_gel.jpg"></p>
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                                <p><small>Gel shows that extraction of w subunit from pWJ66 was successful, but opening of pdCas9 was not.</small></p>
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                        <h2>05.10.2015</h2>
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                            <p><small>2nd attempt to open pdCas9 by PCR done with different annealing temperature and extension times. PCR of w subunit from pWJ66 used as positive control.</small></p>
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                            <h3>Materials and method</h3>
+
 
                             <ul>
 
                             <ul>
                                 <li>20 µl Phusion PCR (cf. Protocols) with 1-3 ng pdCas9 from Miniprep samples 1 and 2 using following parameters:</li>
+
                                 <li>20 µl Phusion PCR (cf. Protocols) with 1 ng pdCas9 testing parameters such as HF vs. GC buffer, annealing temperatures and extension times</li>
                                <ul>
+
                                    <li>A) Annealing temperature (Ta): 56°C; extension time (ext): 2 minutes 30 seconds, 30 cycles, HF buffer</li>
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                                  <li>B) Ta: 59°C, ext: 2 minutes 30 seconds, 30 cycles, HF buffer</li>
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                                    <li>C) Ta: from 66°C to 56°C lowering temperature by 1°C at each cycle, followed by 20 cycles with Ta 56°C, ext: 2 minutes 30 seconds, HF buffer</li>
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                                    <li>D) Same as (C) but with GC buffer</li>
+
                                    <li>E) Ta: 56°C, ext: 3 minutes, 30 cycles, HF buffer</li>
+
                                    <li>F) Ta: 59°C, ext: 3 minutes, 30 cycles HF buffer</li>
+
                                    <li>G) Same as (F) but with GC buffer</li>
+
                                </ul>
+
 
                                 <li>PCR product purification (cf. Protocols)</li>
 
                                 <li>PCR product purification (cf. Protocols)</li>
 
                                 <li>Agarose gel electrophoresis of purified PCR products</li>
 
                                 <li>Agarose gel electrophoresis of purified PCR products</li>
 
                             </ul>
 
                             </ul>
                            <h3>Results</h3>
+
                        <h2>Results</h2>
                                <p align="center"><img src="https://static.igem.org/mediawiki/2015/e/e4/10.06_pcr.jpg"></p>
+
                            <img src="https://static.igem.org/mediawiki/2015/6/6e/10.05_pcr_pdcas9.jpg" align="middle">
 
+
                            <p><small>After testing many parameters, we were able to amplify the plasmid succesfully, as seen on gel above.</br>For further steps sample from lane 1 was used. Its parameters were: HF buffer, annealing temperature 59°C, extension time 2 minutes 30 seconds.</small></p>
 
+
                </div>
 
+
 
+
 
+
 
+
  
 +
    <!-- PCR PWJ66-->
 +
                <div id="PCRpwj66" class="panel">
 +
                    <h1>Assemble pdCas9-w:</br>Extract w subunit from pWJ66 by PCR</h2>
 +
                        <img src="https://static.igem.org/mediawiki/2015/e/ee/7.05_pcr_gel.jpg" align="middle">
 
                 </div>
 
                 </div>
  

Revision as of 14:55, 30 July 2015

E. Coli Laboratory Notebook

Assemble pdCas9-w:
Open pdCas9 by PCR

We received plasmid pdCas9 in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate it.

Materials and method

  • 20 µl Phusion PCR (cf. Protocols) with 1 ng pdCas9 testing parameters such as HF vs. GC buffer, annealing temperatures and extension times
  • PCR product purification (cf. Protocols)
  • Agarose gel electrophoresis of purified PCR products

Results

After testing many parameters, we were able to amplify the plasmid succesfully, as seen on gel above.
For further steps sample from lane 1 was used. Its parameters were: HF buffer, annealing temperature 59°C, extension time 2 minutes 30 seconds.

Assemble pdCas9-w:
Extract w subunit from pWJ66 by PCR

Still under construction