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.