Difference between revisions of "Team:EPF Lausanne/Notebook/Ecoli"
Line 210: | Line 210: | ||
<td> </td> | <td> </td> | ||
<td>72°C</td> | <td>72°C</td> | ||
− | <td><mark>7 minutes</mark></td> | + | <td><mark><small>7 minutes</small></mark></td> |
</tr> | </tr> | ||
<tr> | <tr> |
Revision as of 13:55, 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) 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. 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 will extract the w subunit to fuse it to our own dCas9.
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