Prep of chemically competent E. Coli and transformation (KCM method)

1. Grow up 500 ml of culture of either XL1-blue or DH5α in LB to OD₆₀₀ of 0.4-0.5 (start from a 1 in 100 dilution of a fresh overnight; maintain selective pressure for the episome in the overnight culture in necessary)

2. Swirl the culture flask in an ice bath for 1-2 minutes to pre-chill the cells.

3. Spin down cells (in sterile pre-chilled centrifuge bottles) at 5K for 10 minutes at 4°C.

4. Re-suspend into 1/10 volume (i.e. 50 mls) of ice cold TSS

5. Aliquot 0.5 ml into pre-chilled sterile Eppendorf tubes and snap freeze in liquid nitrogen. Store at -70°C.

1. Slowly thaw an aliquot of cells on ice

2. Set up a second tube containing 20 µl of 5X KCM; add DNA and ddH₂0 up to a total volume of 100 µl. Keep on ice

3. Add 100 µl of the cell suspension to the second tube; mix and keep on ice for 20 minutes.

4. Heat shock the cells by transferring to room temp for 20 minutes OR to 37°C for 5 minutes.

5. Add 1 ml of pre-warmed LB or SOC medium to the tube; incubate at 37°C for 40-60 minutes.

6. Plate out 100 µl of the transformation onto selective medium (LB-amp). Spin down the remaining cells, re-suspend into 100 µl of fresh LB and plate onto a second plate.

       TSS =                         100 ml                   5X   KCM =                    100 ml               10 ml  

         LB                             73 ml                       0.5 M    KCl                 3.73 g            2.5 ml (2 M KCl)

         10% PEG 3350           20 ml (50%)             0.15 M  CaCl₂                   2.21 g            2.5 ml (1 M MgCl₂)

         5% DMSO                  5 ml                        0.25 M  MgCl₂              5.08 g            1.5 ml (1 M CaCl₂)

         20mM MgSO₄                 2 ml  (1 M)                                                                                 H₂0 up to 10 ml

         pH= 6.5