Preparing Electrocompetent Cells

1. Add 2ml of an overnight culture to 500ml of fresh 2xyt media

2. grow to an O.D. of 0.5-0.6

3. Chill cells on ice *Keep cells on Ice from here on.

4. Transfer culture to chilled centrifuge tubes

5. Spin down culture at 4°C for 10 minutes at 5k rpm

6. wash cells with 250ml of ice cold sterile ddH2O.

7. spin again at 4°C for 10 minutes at 5k rpm.

8. Wash cells with 250ml of ice cold sterile ddH2O.

9. Spin again at 4°C for 10 minutes at 5k rpm

10. Wash cells with 20ml of filter sterilized* 10% glycerol

11. spin cells at 4°C

12. resuspend cells in ice cold 10% glycerol. Use approximately same volume as the cell pellet.

13. If using cells directly aliquot 100ul to eppendorf tubes on ice. Use for electroporation.

14. if cells will not be used directly aliquot 100ul to eppendorf tubes on dry ice, and store at -70°C. Thaw on ice when needed.