Difference between revisions of "Team:Paris Saclay/Notebook/September/19"

Revision as of 10:06, 19 September 2016

Contents

1 Monday 19^th September
- 1.1 Lab work
  - 1.1.1 Visualization
    - 1.1.1.1 PCR of pSB1C3 from dCas9 ST - GFP 11 clone 8 to correct mutations

Monday 19^th September

Lab work

Visualization

PCR of pSB1C3 from dCas9 ST - GFP 11 clone 8 to correct mutations

By Maxence

In order to correct mutations in dCas9 ST - GFP 11, PCR was performed with the following protocol.

For each 50μl of reaction, mix the following reagents :

1 µL of matrix
1 µL of dNTPs (10mM)
2.5 µL of each primer mix (10µM)
10 µL of buffer HF (5X)
0,5 µL of Phusion polymerase
32.5 µL of nuclease free water

Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice. Perform PCR as follow:

Step	Temperature	Time
Initial denaturation	98°C	30sec
30 cycles	98°C	10sec
	72°C	30sec
	72°C	t
Final Extension	72°C	5min
Hold	4°C	$\infty$

Primers used were:

Matrix	dCas9 ST - GFP 11 clone 8	dCas9 ST - GFP 11 clone 8
Primers	iPS174 and iPS175	iPS173 and iPS176
Tm	72°C	72°C
t	3min	50sec