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        ELECTROPORATING E. COLI

        互联网

        2077

        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℃

        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

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