Team:Pasteur Paris/Week 1
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WEEK 1
Materials :
Protocol : 1/ Thaw on ice one tube of DH5α cells. Place 1.5mL microcentrifuge tubes on wet ice. 2/ Gently mix cells with the pipette tip and aliquot 50µL for each transformation into a 1.5ml centrifuge tube. 3/ Refreeze any unused cells. 4/ Add 1 to 5 µL (1-10 ng) DNA to the cell and mix gently (Not mix by pupetting up and down) 5/ Incubate tubes on ice for 30 min 6/ Heat shock cells for 40s in a 42°C water bath without shaking. 7/ Place tube on ice for 3 minutes. 8/ Add pre-warmed 700µL SOC medium (XXXXXXXX). 9/ Incubate at 37°C for 40 min under shaking condition (200 rpm) 10/ Spread 200 µL of transformed cell on LB + Cm34 (chloramphenicol 34µg/mL). 11/ Incubate dished overnight at 37°C 05-06-15 : Bacterial amplification : xxxxxxxxxxx Materials : • Petri dishes • LB with or without chlroramphénicol • 15mL falcon tubes (3 per Petridish used) Protocol : 1/ Fill the tubes with 5mL of the correct media (3 tubes per strain) 2/ Pick three transformed clones per Petri box (1 clone per 15mL tube) 3/ Incubate in agitation (8 hours at 200 rpm, 37°C) ^ |