Team:Aix-Marseille/Protocol:electrocompetent ecoli cells
From 2014.igem.org
Electrocompetent Escherichia coli 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. Subsequent resuspensions may be done in the same bottle. Cells must remain cold for the rest of the procedure: Transport tubes on ice and resuspend on ice in the cold room.
- 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.
Preparation of competent cells
Transformation