Team:Paris/August 11

Transformation

• Protocol for Top 10 cells : (see # 13)

Digestion

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_138), 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 :

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