Team:Paris Saclay/Notebook/September/16

Friday 16th September

Lab work

Visualization

Colony PCR of 16 clones containing FRB - GFP 11 in pSB1C3

By Maxence & Mahnaz

Colonies were obtained for the Gibson done the 7th September but not for the one done the 8th September. A colony PCR was done for 16 clones from the 7th September. For that purpose, 16 were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.

For each clones contained in 20 μl water, 4.13 μL of the following mix were added :

  • 2.5 µL DreamTaq Buffer
  • 0.5 µL of dNTPs (10mM)
  • 1 µL of each primer mix (10µM)
  • 0.13 μl of DreamTaq Pol

PCR was performed as follow:

Step Temperature Time
Initial denaturation 95°C 3 min
30 cycles 95°C 30 sec
61°C 30 sec
72°C 1 min 30 sec
Final Extension 72°C 7 min
Hold 4°C $\infinity\$ 
Primers used were:
Matrix Clones containing FRB - GFP 11 in pSB1C3
Primers iPS83 and iPS84

After amplification, 3 µL of each PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

PCR products expected were :

PCR products Expected band size (bp)
FRB - GFP 11 in pSB1C3 730

GEL 6

All PCR products were at the good size and seemed to be concentrated. It could be explained as these clones were grown for 2 days. Clones 4, 9, 13 and 16 were selected and were grown at 37°C overnight.





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