Team:EPF Lausanne/Notebook/Ecoli
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.
