WangLab:Electro-competent

The Preparation of E. coli (BJ5183) by Electroporation(06-24-2005 wyy)

1. Place the competent cells on LB plate, 37°C overnight (16-20h).
2. Pick a single bacterial colony from a plate that has been incubated for 16-20 hours at 37°C. Transfer the colony into 20 ml of LB medium in a 100-ml flask. Incubate the culture overnight at 37°C with vigorous aeration (250 rpm in a rotary shaker).
3. Inoculate two aliquots of 250 ml of prewarmed LB medium in separate 1-liter flasks with 5 ml of the overnight bacterial culture. Incubate the flasks at 37°C with agitation (300 cycles/minute in a rotary shaker). Measure the OD600 of the growing bacterial cultures every 20 minutes.
4. When the OD600 of the cultures reaches 0.4, rapidly transfer the flasks to an ice-water bath for 15-30 minutes. Swirl the culture occasionally to ensure that cooling occurs evenly. In preparation for the next step, place the centrifuge bottles in an ice-water bath.
5. Transfer the cultures to ice-cold centrifuge bottles. Harvest the cells by centrifugation at 1000g (2500 rpm in a Sorvall GSA rotor) for 15 minutes at 4°C. Decant the supernatant and resuspend the cell pellet in 250 ml of ice-cold pure H2O.
6. Harvest the cells by centrifugation at 1000g (2500 rpm in a Sorvall GSA rotor) for 20 minutes at 4°C. Decant the supernatant and resuspend the cell pellet in125 ml of ice-cold 10% glycerol.
7. Harvest the cells by centrifugation at 1000g (2500 rpm in a Sorvall GSA rotor) for 20 minutes at 4°C. Decant the supernatant and resuspend the pellet in 5 ml of ice-cold 10% glycerol.
8. Harvest cells by centrifugation at 1000g (2500 rpm in a Sorvall GSA rotor) for 20 minutes at 4°C. Carefully decant the supernatant and use a Pasteur pipette attached to a vacuum line to remove any remaining drops of buffer. Resuspend the pellet in 1 ml of ice-cold 10% glycerol.
9. Measure the OD600 of a 1:100 dilution of the cell suspension. Dilute the cell suspension to a concentration of 2 x 1010 to 3 x 1010 cells/ml (1.0 OD600 = approx. 2.5 x 108 cells/ml) with ice-cold 10% glycerol.
10. Transfer 40 µl of the suspension to an ice-cold electroporation cuvette (0.2-cm gap) and test whether arcing occurs when an electrical discharge is applied . If so, wash the remainder of the cell suspension once more with ice-cold 10% glycerol to ensure that the conductivity of the bacterial suspension is sufficiently low (<5 mEq).
11. Store the cells at -70°C until required. For storage, dispense 40-µl aliquots of the cell suspension into sterile, ice-cold 0.5-ml microfuge tubes, drop into a bath of liquid nitrogen, and transfer to a -70°C freezer.