Team:Paris/August 7

Result of the isolation of colonies

E0240 and pSB3K3 are ok : there is a lot of single colonies S120 and S121 : there is a problem, there is nothing on the plates. We have to check whether those strains are really resistant to Amp.

Results of the PCR we did last night

PCR with gradient to amplify pflhDC

Standart PCR to amplify pflgA, pflgB and pflhB

Transformations

Protocol

Use of TOP10 chemically competentcells

• Defroze competent cells on ice during 5'
• Add 5µl of DNA Ligation in 50µL of competent bacterias (or 1µL for the positive control puc19)
• Incubate 30' on ice
• Heat-shock the cells during 30" at 42°C without shaking
• Put 2' on ice
• Add 250µL of pre-warmed SOC medium (4°C)
• Incubate 1h at 37°C under shaking (225rpm)
• Spin at 5.000rpm during 30"
• Remove 150µL of supernatant
• Resuspent the pellet in the 150µL left
• Spread on adequated plates
• Incubate O/N at 37°C

List of the Ligation Transformation