Difference between revisions of "Team:EPF Lausanne/Notebook/Ecoli"
Line 149: | Line 149: | ||
<nav id="affix-nav" class="sidebar hidden-sm hidden-xs"> | <nav id="affix-nav" class="sidebar hidden-sm hidden-xs"> | ||
<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=" | + | <li class="active"><a href="#">Assemble pdCas9-w</a></li> |
− | + | <ul> | |
− | + | <li><a href="#PCRpdcas9">Open pdCas9 by PCR</a></li> | |
− | + | <li><a href="#PCRpwj66">Extract w subunit from pWJ66 by PCR</a></li> | |
− | + | </ul> | |
− | + | <li><a href="#">Assemble pdCas9-w-sgRNAs</a></li> | |
− | </li> | + | <li><a href="#">Assemble pWJ89alt1</a></li> |
+ | |||
</ul> | </ul> | ||
</nav> | </nav> |
Revision as of 15:46, 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.
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.
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.