Team:Aix-Marseille/Protocol:electrocompetent ecoli cells

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Electrocompetent Escherichia coli cells

Preparation of competent cells

Streak E.coli cells (W3110) on an LB plate with or without antibiotics.
Allow cells to grow at 37°C overnight.
Place three or four colonies in 5 mL LB media (+ antibiotic selection if necessary), grow overnight at 37°C.
Dilute the preculture to OD600 = 0,005 in 100 mL LB.
Allow cell to grow at 37°C (250 rpm), until OD600= 0.4 (about 2-3 hours).
Transfer cells to 2 centrifuge bottles (50 mL), and place cells on ice for 20 mins.
Centrifuge cells in Sorval GSA rotor at 4°C for 10 mins at 5 000 tr/min.
Discard the supernatant and resuspend cells in 1 Vol ice-cold sterile 10% glycerol (gentle shaking on ice or in the coldroom is the best way to go). Spin the cells at 6000 tr/min at 4°C for 10 mins.
Resuspend the pellet in 0.5 Vol of ice-cold sterile 10% glycerol. Spin at 8000 rpm at 4°C for 15 mins (after washing, the cells become harder and harder to pellet down).
Wash the cells with ~20mL (per liter of the original cell culture) of ice-cold sterile 10% glycerol and transfer the cell suspension to a pre-chilled small centriguge tube. Spin the cells at 10000 tr/min at 4°C for 15 mins.
Resuspend the cells in 2-3 ml of ice-cold sterile 10% glycerol per liter of the original cell culture.
Aliquot the cells into pre-chilled microfuge tubes (40 µl per transformation in a 0.1-0.2 cm gap electroporation cuvette). The cells are now ready to use. For long-term storage, store at -80°C the tubes with 50 µl aliquots.

To use the frozen cells, thaw on ice the individual aliquots, add salt-free DNA to them (typically 1-5 µl), mix by pipetting, and transfer the mix to a pre-chilled electroporation cuvette. Electroporate the cells according to manufacturer's instructions.
Incubate the mixture on ice for 35 minutes.
Immediately after the pulse, add 1ml of room temperature liquid LB or SOC media to the cells, and recover them by shaking the samples at 37°C for 1h.
Plate the cells on LB plates supplemented with an appropriate selective agent, allow the excess liquid to dry/absorb into the plate. Incubate the plate up-side-down overnight at 37°C.