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.
Materials and method
- 20 µl Phusion PCR (cf. Protocols) with 1-3 ng pdCas9 from Miniprep samples 1 and 2 using following parameters:
- A) Annealing temperature (Ta): 56°C; extension time (ext): 2 minutes 30 seconds, 30 cycles, HF buffer
- B) Ta: 59°C, ext: 2 minutes 30 seconds, 30 cycles, HF buffer
- C) Ta: from 66°C to 56°C lowering temperature by 1°C at each cycle, followed by 20 cycles with Ta 56°C, ext: 2 minutes 30 seconds, HF buffer
- D) Same as (C) but with GC buffer
- E) Ta: 56°C, ext: 3 minutes, 30 cycles, HF buffer
- F) Ta: 59°C, ext: 3 minutes, 30 cycles HF buffer
- G) Same as (F) but with GC buffer
- PCR product purification (cf. Protocols)
- Agarose gel electrophoresis of purified PCR products
Results
