Difference between revisions of "Team:EPF Lausanne/Notebook/Ecoli"
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<h2>Materials and method</h2> | <h2>Materials and method</h2> | ||
<ul> | <ul> | ||
− | <li>20 µl Phusion PCR (cf. Protocols) | + | <li>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</li> |
<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> | ||
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<h2>Materials and method</h2> | <h2>Materials and method</h2> | ||
<ul> | <ul> | ||
− | <li>20 µl Phusion PCR (cf. Protocols) with 1 ng pWJ66 using HF buffer and following thermocycling settings:</li> | + | <li>20 µl Phusion PCR (cf. Protocols) with 1 ng pWJ66 using appropraite primers, HF buffer and following thermocycling settings:</li> |
<table width="70%" align="center"> | <table width="70%" align="center"> | ||
<tr> | <tr> | ||
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<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"> | ||
− | <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> | ||
Revision as of 15:38, 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.