Team:Paris Saclay/Notebook/September/16

Friday 16th 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