Electrocompetent E.Coli

Dominik Haudenschild, December 4, 1993 at CBRC

Prepare the cells

1. Inoculate a single colony of E. coli cells into 5 ml LB medium. Grow 5 hr to overnight at 37°C with moderate shaking

2. Inoculate 2.5 ml of the culture into 500 ml LB medium in a sterile 2-liter flask. Grow at 37°C, shaking at 300 rpm, to an OD600 of ~0.5 to 0.7.

Best results are obtained by harvesting cells at an OD600 of ~0.5 to 0.6.

3. Chill cells in an ice-water bath 10 to 15 min and transfer to a prechilled 1-liter centrifuge bottle.

Cells should be kept at 2°C for all subsequent steps.

4. Centrifuge cells 10 min, 2500g at 2°C.

5. Pour off supernatant and resuspend the pellet in 5 ml ice-cold water. Add 500 ml ice-cold 10% Glycerol and mix well. Centrifuge cells as in step 4.

6. Pour off supernatant immediately and resuspend the pellet by swirling in remaining liquid.

7. Add another 500 ml ice-cold 10% Glycerol, mix well, and centrifuge again as in step 4.

8. Pour off supernatant immediately and resuspend the pellet by swirling in remaining liquid.

9b. If frozen cells are to be used for electroporation, add 40 ml ice-cold 10% glycerol to the cells and mix well. Centrifuge cells 2500g 5 minutes.

Estimate the pellet volume and add an equal volume of ice-cold 10% glycerol to resuspend cells (on ice). Place 100-ul aliquots of cells into prechilled microcentrifuge tubes and freeze on dry ice (not in liquid nitrogen). Store at -80°C.

Note: Cells in water are not very stable, and gentle manipulation is required. Pipet slowly!