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        Rubidium Chloride method for Transformation Competent E. coli

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        1016

         

        Rubidium Chloride method for Transformation Competent E. coli

        Procedure

        1. Inoculate 1 ml from overnight culture into 100 ml Psi broth (scale up or down as needed). Incubate at 37 C with aeration to A550=0.48

        2. Ice 15 min.

        3. Pellet cells in appropriate centrifuge tube 3-5000 x g 5 min (~5000 rpm in a Sorvall SS-34 rotor)

        4. Discard supernatant and add 0.4 volume (ie of original volume, here it is 40 ml) TfbI, resupend and ice 15 min.

        5. Pellet cells as in #3.

        6. Discard supernatant and resupend in 0.04 volume TfbII, ice 15 min and either use immediately or quick freeze at -70C for storage. I usually save these in 0.25 to 0.5 ml aliquots. Quick freeze in ethanol-dry ice or liquid nitrogen prior to storage in a -70 to -80 C freezer. Thaw on ice just before using in a transformation experiment.

        I typically transform 50 ul cells with 2-10 ul of a ligation reaction, and you should get between 1x10exp8 to 1x10exp9 cfu's/ug DNA.


        Medium and Buffers

        Psi broth (per liter)
        compound amount
        Bacto yeast extract 5 g
        Bacto Tryptone 20 g
        magnesium sulfate 5 g
        pH 7.6 with potassium hydroxide

         

        TfbI (per 200 ml)
        compound amount final molarity/conc.
        potassium acetate .588 g 30 mM
        rubidium chloride 2.42 g 100 mM
        calcium chloride 0.294 g 10 mM
        manganese chloride 2.0 g 50 mM
        glycerol 30 ml 15% v/v
        pH 5.8 with dilute acetic acid

         

        TfbII (per 100 ml)
        compound amount final molarity/conc.
        MOPS 0.21 g 10 mM
        calcium chloride 1.1 g 75 mM
        rubidium chloride 0.121 g 10 mM
        glycerol 15 ml 15% v/v
        pH 6.5 with dilute NaOH

         

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