Team:Paris Saclay/Notebook/October/3

Monday 3rd October

Lab work

Visualization

Colony PCR of X clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021)

By Maxence

For that purpose, X clones 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, 5.13 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
48.4°C 30 sec
72°C 30sec
Final Extension 72°C 7 min
Hold 4°C $\infty$

Primers used were:

Matrix Clones containing FKBP - GFP 10 in pSB1C3 (pPS16_19)
Primers iPS168 and iPS169

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)
FKBP - GFP 10 in pSB1C3 (pPS16_019) 1030