Team:Paris Saclay/Notebook/October/14

Thursday 14th October

Visualization

PCR of GFP 1.9 in pUC19 (pPS16_009) and GFP 1.9 in gblock

By Sylvie

A new PCR was run in order to obtain a new GFP 1.9 PCR product. For each amplification, two matrix were used: GFP 1.9 in pUC19 (pPS16_009) and GFP 1.9 in gblock.

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

  • 1 µL of matrix (diluted 10X)
  • 1 µL of dNTPs (10mM)
  • 2.5 µL of each primer mix (10µM)
  • 10 µL of buffer (5X)
  • 0,5 µL of Phusion polymerase
  • 30 µL of nuclease free water
  • 1.5 µL of DMSO

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
60°C 30sec
72°C 30 sec
Final Extension 72°C 5min
Hold 4°C $\infty$

Primers used were:

Matrix GFP 1.9 in pUC19 (pPS16_009) extracted the 2nd October
Primers iPS84 and iPS140

5 µL of PCR products and 20 µL of DNA ladder were placed in wells and migrated at 100V during 30 min, in odrder to verify the DNA quantity.

PCR products expected were :

PCR products Expected band size (bp)
GFP 1.9 862
Result of the migration