Difference between revisions of "Team:Ionis Paris/Protocol 5"
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Latest revision as of 19:51, 19 October 2016
Inoculate 100µL of cells in 100mL LB medium
Put 1.11g of CaCl2 in 100mL of sterile H2O to obtain a final concentration of 0.1M of CaCl2 (Molar mass of Cacl2: 110,98g/mol)
Inoculate 3mL of the overnight culture cell from in 90mL of LB. Shake at 37°C until the DO is around 0,6
Protocol 5 : Preparation of competent cells
Aim: Prepare competent bacteria for subsequent transformation
Inoculation
Let the culture grow O/N at 37°C
CaCl2 preparation
Competency
NB : all the following steps must be perform in ice
Transfer cells into sterile ice-cold Falcon tubes
Incubate on ice for 30 min
Spin cells at 4000 rpm for 10 min at 6°C
Discard supernatant and try to drain all remaining media
Gently resuspend in 10mL cold 0,1M CaCl2
Incubate on ice 20 min
Centrifuge 10 min at 4000 rpm at 4°C
Discard supernatant and vortex, gently resuspend on 5mL cold 0,1M CaCl2/15% Glycerol
Transfer in 2mL cryotubes
Store at -80°C for subsequent transformation
