Team:Paris Bettencourt/Protocols/Electrocompetent Cells Preparation
Ferment It Yourself
iGEM Paris-Bettencourt 2O15
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Notebook
Vitamin A Vitamin B2 Vitamin B12 Phytase Riboswitch Differentiation on E. coli Differentiation on S. cerevisiae Manufacturing Idli and Micro-organisms Electrocompetent Cells Preparation Protocol - Inoculate two 250mL LB flasks with 100µL of an overnight culture of DH5α
- incubate until the the DO600 reach 0.5 to 0.7
- place the cultures on ice for 15 minutes
- pour the culture in cold sterile 50mL falcon tubes
- centrifuge them for 10 minutes at 6000rpm
- throw the supernatant
- resuspend the cells in 50mL cold distilled water
- centrifuge them for 10 minutes at 6000rpm
- throw the supernatant
- resuspend the cells in 25mL cold distilled water
- centrifuge them for 10 minutes at 6000rpm
- throw the supernatant
- resuspend the cells in 12.5mL cold 10% glycerol
- centrifuge them for 10 minutes at 6000rpm
- throw the supernatant
- resuspend the cells in 5mL cold 10% glycerol
- make aliquots of the desire volume in microcentrifuge tubes and freeze them at -80°C