Difference between revisions of "Team:EPF Lausanne/Notebook/Ecoli"
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<li><a href="#PCRpdcas9">Open pdCas9 by PCR</a></li> | <li><a href="#PCRpdcas9">Open pdCas9 by PCR</a></li> | ||
<li><a href="#PCRpwj66">Extract w subunit from pWJ66 by PCR</a></li> | <li><a href="#PCRpwj66">Extract w subunit from pWJ66 by PCR</a></li> | ||
+ | <li><a href="#gibsonpdcas9w">Gibson assembly of pdCas9-w</a></li> | ||
</ul> | </ul> | ||
<li><a href="#">Assemble pdCas9-w-sgRNAs</a></li> | <li><a href="#">Assemble pdCas9-w-sgRNAs</a></li> | ||
<li><a href="#">Assemble pWJ89alt1</a></li> | <li><a href="#">Assemble pWJ89alt1</a></li> | ||
− | + | ||
+ | |||
</ul> | </ul> | ||
</nav> | </nav> | ||
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<div id="PCRpdcas9" class="panel"> | <div id="PCRpdcas9" class="panel"> | ||
<h1>Assemble pdCas9-w: Open pdCas9 by PCR</h2> | <h1>Assemble pdCas9-w: Open pdCas9 by PCR</h2> | ||
− | <p><small>We received plasmid pdCas9 in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate it.</br>pdCas9 is a plasmid containing dCas9 under a Tetracyclin inducible promoter and a Chloramphenicol resistance gene, available on Addgene under the name pdCas9-bacteria.</small></p> | + | <p><small>We received plasmid pdCas9 in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate it.</br>pdCas9 is a plasmid containing dCas9 under a Tetracyclin inducible promoter and a Chloramphenicol resistance gene, available on Addgene under the name pdCas9-bacteria.</br>We opened this plasmid by PCR to be able use later on in a Gibson assembly.</small></p> |
<h2>Materials and method</h2> | <h2>Materials and method</h2> | ||
<ul> | <ul> | ||
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</ul> | </ul> | ||
<h2>Results</h2> | <h2>Results</h2> | ||
− | <img src="https://static.igem.org/mediawiki/2015/archive/6/6e/20150804142724%2110.05_pcr_pdcas9.jpg" | + | <img src="https://static.igem.org/mediawiki/2015/archive/6/6e/20150804142724%2110.05_pcr_pdcas9.jpg" style="width:250px;height:158px;"> |
<p><small>Linearized pdCas9-w is expected to be 6705 bp.</br>After testing many parameters, we were able to linearize the plasmid succesfully, as seen on gel above.</br>For further uses, sample from lane 1 was used. It was prepared with HF buffer and the following thermocycling settings:</small></p> | <p><small>Linearized pdCas9-w is expected to be 6705 bp.</br>After testing many parameters, we were able to linearize the plasmid succesfully, as seen on gel above.</br>For further uses, sample from lane 1 was used. It was prepared with HF buffer and the following thermocycling settings:</small></p> | ||
<table width="70%" align="center"> | <table width="70%" align="center"> | ||
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<div id="PCRpwj66" class="panel"> | <div id="PCRpwj66" class="panel"> | ||
<h1>Assemble pdCas9-w: Extract w subunit from pWJ66 by PCR</h1> | <h1>Assemble pdCas9-w: Extract w subunit from pWJ66 by PCR</h1> | ||
− | <p><small>We received plasmid pWJ66 in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate it.</br>pWJ66 is a plasmid containing dCas9 fused at its C-terminal to the w subunit of RNA polymerase as well as a tracrRNA gene and a CRISPR array gene, available on Addgene. We extracted the w subunit to fuse it to our own dCas9.</small></p> | + | <p><small>We received plasmid pWJ66 in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate it.</br>pWJ66 is a plasmid containing dCas9 fused at its C-terminal to the w subunit of RNA polymerase as well as a tracrRNA gene and a CRISPR array gene, available on Addgene.</br>We extracted the w subunit to fuse it to our own dCas9 by Gibson assembly.</small></p> |
<h2>Materials and method</h2> | <h2>Materials and method</h2> | ||
<ul> | <ul> | ||
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</ul> | </ul> | ||
<h2>Results</h2> | <h2>Results</h2> | ||
− | <img src="https://static.igem.org/mediawiki/2015/archive/e/ee/20150804153054%217.05_pcr_gel.jpg"> | + | <img src="https://static.igem.org/mediawiki/2015/archive/e/ee/20150804153054%217.05_pcr_gel.jpg" style="width:250px;height:374px;"> |
<p><small>Successful PCR reactions are expected to yield 340 bp fragments.</br>As seen on gel, PCR was succesful for sample in lane 1.</small></p> | <p><small>Successful PCR reactions are expected to yield 340 bp fragments.</br>As seen on gel, PCR was succesful for sample in lane 1.</small></p> | ||
</div> | </div> | ||
+ | |||
+ | <!-- GIBSON PDCAS9-W --> | ||
+ | <div id="gibsonpdcas9w" class="panel"> | ||
+ | <h1>Assemble pdCas9-w: Gibson assembly of pdCas9-w</h1> | ||
+ | <p><small>Using PCR products from experiments described above, we fused the w subunit of DNA polymerase to dCas9 by Gibson assembly.</small></p> | ||
+ | <h2>Materials and method</h2> | ||
+ | <ul> | ||
+ | <li>Gibson assembly (cf. Protocols) with purified PCR products:</li> | ||
+ | <ul> | ||
+ | <li>Open pdCas9: 0.02 pmol = 95 ng</li> | ||
+ | <li>w subunit extracted from pWJ66: 0.06 pmol = 12.5 ng</li> | ||
+ | </ul> | ||
+ | |||
− | |||
− | |||
Revision as of 16:43, 4 August 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.pdCas9 is a plasmid containing dCas9 under a Tetracyclin inducible promoter and a Chloramphenicol resistance gene, available on Addgene under the name pdCas9-bacteria.We opened this plasmid by PCR to be able use later on in a Gibson assembly.
Materials and method
- 20 µl Phusion PCR (cf. Protocols) of 1 ng pdCas9 with appropriate primers, 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
Linearized pdCas9-w is expected to be 6705 bp.After testing many parameters, we were able to linearize the plasmid succesfully, as seen on gel above.For further uses, sample from lane 1 was used. It was prepared with HF buffer and the following thermocycling settings:
Step | Temperature | Time | |
---|---|---|---|
Initial Denaturation | 98°C | 40 seconds | |
25 cycles | Denaturation | 98°C | 15 seconds |
Annealing | 59°C | 22 seconds | |
Extension | 72°C | 2 minutes 30 seconds | |
Final Extension | 72°C | 7 minutes | |
Hold | 4°C |
Assemble pdCas9-w: Extract w subunit from pWJ66 by PCR
We received plasmid pWJ66 in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate it.pWJ66 is a plasmid containing dCas9 fused at its C-terminal to the w subunit of RNA polymerase as well as a tracrRNA gene and a CRISPR array gene, available on Addgene.We extracted the w subunit to fuse it to our own dCas9 by Gibson assembly.
Materials and method
- 20 µl Phusion PCR (cf. Protocols) with 1 ng pWJ66 using appropraite primers, HF buffer and following thermocycling settings:
- PCR product purification (cf. Protocols)
- Agarose gel electrophoresis of purified PCR products
Step | Temperature | Time | |
---|---|---|---|
Initial Denaturation | 98°C | 30 seconds | |
30 cycles | Denaturation | 98°C | 10 seconds |
Annealing | 62°C | 15 seconds | |
Extension | 72°C | 15 seconds | |
Final Extension | 72°C | 7 minutes | |
Hold | 4°C |
Results
Successful PCR reactions are expected to yield 340 bp fragments.As seen on gel, PCR was succesful for sample in lane 1.
Assemble pdCas9-w: Gibson assembly of pdCas9-w
Using PCR products from experiments described above, we fused the w subunit of DNA polymerase to dCas9 by Gibson assembly.
Materials and method
- Gibson assembly (cf. Protocols) with purified PCR products:
- Open pdCas9: 0.02 pmol = 95 ng
- w subunit extracted from pWJ66: 0.06 pmol = 12.5 ng