Team:Paris/August 11

Transformation

All the ligations were transformed according transformation for Top10 protocol

PCR

We performed PCR on to amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.

PCR amplification

Protocol

• List of Oligos :

• Preparation of the templates :
  Resuspension of 1 colony in 100µl of water.

• Preparation of PCR mix :

For each sample,

1 µl dNTP
10 µl Buffer Phusion 5x
2,5 µl Oligo_F
2,5 µl Oligo_R
1µl template
1 µl Phusion
50 µl qsp H2O (33µl)

• Program PCR_Screening : Annealing 30" at 60°C - Time élongation 1'30" at 72°C - Number cycle : 30

PCR verification/Analysis

Analysis of PCR product (Gel 1)

Analysis of PCR product (Gel 2)

After the PCR :

• 2*3µl have been analysed by electrophoresis
• the other 44µl of PCR products have been purified by the Promega kit.

• Electrophoresis

ladder : 10µl ladder 1 kb
samples : 3µl of PCR products + 2µl of Loading Dye
migration 30min at 100V, on a 1% agarose gel

• Results :

==> Conclusion : We observed the size expected for the PCR products, but not for pflhDC (PCR_134), is right. We hypothesis for PCR_138 that the size is longer that expected due to the aspecific fixation of Oligo O111 (upper to the real site).

• Electrophoresis

Analysis of PCR product (Gel 3)

ladder : 10µl ladder 100 bp
samples : 3µl of PCR products + 2µl of Loading Dye
migration 30min at 100V, on a 2% agarose gel

• Results :

==> Conclusion : We need to repeat the experiments.

Culture of ligation transformants

• 4 clones of each transformation were cultured in 7,5 mL LB + ampicilline. The colonies picked up were the rest of those already picked up from the transformation plate (for the PCR screening of August 8).
• 37°C overnight