ELECTROPORATING E. COLI

1.O/N (5ml E.coli in LB^-->1ml into 200ml LB/37℃

2.Grow 2-3H to A660 ~0.3-0.4

3.Chill cells 10'/ice Spin down 10',5K,4℃

4.Wash 1X 200ml 10% glycerol (sterile)40℃; Spin down 10',5K,4℃

5.Wash 1X 40 ml 10% glycerol^--> transfer to S34 tubes & spin down 5',5K,4℃

**wash means complete resuspention to remove all salt

6.Invert tube to drain SN ~5sec (soft plt).with p200,resuspend cells in remaining SN & transfer to eppendorf.Wash SS34 tube with another 100μl 10% glycerol and pool with cells.Vf ~300-500μl.Use ~ 50μl cells/electroporation.

7.Turn on machine (back)-->set 2.5kV,25 uF.Pulse controller to 200 ohms (wide 0.2cm cuvette)

8.1-2μl ligation/ 50μl cells (eppendorf).Chill cuvettes.

9.Transfer cells & DNA to cold 0.2cm cuvette

10.IMMEDIATELY electroporate @ 2.5kV,25 uF,200 ohms (expect time constant 3.5-5msec)

11.IMMEDIATELY add 1ml cold SOC.Transfer to eppendorf.

12.37℃/60'

13.Plate 50-200μl cells (50 ~ 200-300 colonies)

Dr.DE Koshland,Carnegie Institution of Washington

http://www.ciwemb.edu/labs/koshland/Protocols/BACTERIA/electroporate.html