Team:EPF Lausanne/Notebook/Ecoli
E. Coli Laboratory Notebook
Make pdCas9-w:Open pdCas9 and extract w subunit from pWJ66 by PCR
We received plasmids pdCas9 and pWJ66 in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate them.
05.06.2015
Materials and method
- 20 µl Phusion PCR (cf. Protocols) with 1 ng pdCas9/pWJ66, HF buffer, corresponding primers, 30 cycles, annealing temperature 58°C/62°C and extension time 150/15 seconds
- PCR product purification (cf. Protocols)
- Agarose gel electrophoresis of purified PCR products
Results
Gel shows that extraction of w subunit from pWJ66 was successful, but opening of pdCas9 was not.
05.10.2015
2nd attempt to open pdCas9 by PCR done with different annealing temperature and extension times. PCR of w subunit from pWJ66 used as positive control.