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.

Protocol

 * List of Oligos :


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

For each sample,
 * Preparation of PCR mix :

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
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).

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


 * Results :

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

Culture of ligation transformants (pFlgA, pFlgB and pFlhB)

 * 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