Quick & dirty electrocompetent cells preparation FOR TRANSFORMATION OF SUPERCOILED PLASMIDS INTO E. COLI
Before starting, put centrifugation bottles, Falcon tubes and ddH2O at 4°C. Also cool the centrifuges.
1) Inoculate a 1.5 ml culture in LB or appropriated medium and grow overnight at the appropriated temperature in a 12ml red cap tube 2) Quick chill the culture on ice and transfer the cells into a 1.5 mL microcentrifuge tube 3) Harvest the cells at 14000 rpm at 4°C for 45 seconds. 4) Discard the supernatant and resuspend the bacteria in 1 volume of cold sterile ddH2O. 5) Spin at 14000 rpm at 4°C for 45 seconds. 6) Repeat steps 4 and 5 7) Resuspend it in 5µL cold sterile ddH2O.
7) Resuspend in 1 volume of ice-cold 10% glycerol. Glycerol washes are meant for competent cells that will be frozen. 8) Spin the bacteria at 4°C for 10 minutes ...
MgCl2·6H2O. 10 mM. (2 gr.L-1). Glycerol. 10%. (100 mL.L-1). Adjust pH DOWN to 6.4 with 0.1N HCl. Filter-sterilize and store at 4°C. Volumes: Starting. 1st wash.
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