Team:Aix-Marseille/Protocol:electrocompetent ecoli cells

Electrocompetent Escherichia coli cells

Preparation of competent cells

Streak E.coli cells 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 rpm.
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 rpm 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 on ice. Avoid introducing air bubbles into the cell mix in the cuvette as this can cause arcing during electroporation.
Activate electroporator by pressing the ON/STDBY key. For BioRad system, set on EC1.
Bring the ice bucket with cuvettes in it, plus SOC and P1000 pipette close to the electroporator.
Remove the electroporation cuvette containing the competent cells/DNA mix from the ice, wipe off any excess ice/moisture from the cuvette and place it into the electroporator cuvette holder (the cuvettes have a notch protruding from them and as such can only fit into the cuvette holder in one direction).
Slide the cuvette holder all the way back until it is flush with the front of the unit.
Press the PULSE key. A “beep” will sound indicating that electroporation has occurred.
Immediately remove the cuvette and add 650 µL SOC (or LB if you are out of SOC).
Outgrow (recover) the transformed cells by gentle shaking at 37°C for ~1h.
Plate a serial dilution of the transformed cells on selective medium.