Team:Paris Saclay/Notebook/September/9

Friday 9th September

Lab work

Visualization

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

Maxence

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
Tm 30 sec
72°C t
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
Tm 61°C
t 1 min 30

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 7, 9, 15 and 16 were selected and were grown at 37°C overnight.

PCR on GFP 1.9 in pUC19, gblock 1.1, gblock 1.2, gblock 2.1 and gblock 2.2 with 3% DMSO

By Maxence

As the annealing temperature (Tm) seems too high to obtain good results for these amplifications, DMSO was used in order to reduce the annealing temperature during the PCR. For that purpose, PCR was performed on plasmids with the following protocol.For each amplification, two matrix were used: one was a previous PCR products and one was a plasmid.

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 (5X)
  • 0,5 µL of Phusion polymerase
  • 31 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 2min
Hold 4°C $\infty$ 
Primers used were:
Matrix PCR product of gblock 1.1 from 2nd September (Q5) gblock 1.1 9/08 PCR product of gblock 1.2 from 2nd September (Q5) gblock 1.2 11 PCR product of gblock 2.1 from 2nd September (Q5) gblock 2.1 PPS16003 PCR product of gblock 2.2 from 2nd September (Q5) gblock 2.2 PPS16004 PCR product of GFP 1.9 from 8th September GFP 1.9 in pUC19
Primers iPS140 and iPS120 iPS140 and iPS120 iPS121 and iPS122 iPS121 and iPS122 iPS123 and iPS124 iPS123 and iPS124 iPS125 and iPS84 iPS125 and iPS84 iPS84 and iPS140 iPS84 and iPS140

PCR Clean-up of PCR products

By Maxence

PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.

Gel of cleaned up PCR products

By Maxence

After amplification, 3 µL of each cleaned up 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)
gblock 1.1 960
gblock 1.2 960
gblock 2.1 1023
gblock 2.2 808
GFP 1.9 862

GEL ? : FKBP

All PCR products were at the good size.