E. coli Competent Cells

Preparation of competent bacterial cells

1. Strike out stock of cells on LB plates and grow overnight at 37˚C.

2. Pick a single colony from the plate and grow in 5 mL of LB at 37˚C.

3. Inoculate a 500 mL culture with the overnight culture and place it in the shaker at 37˚C until it reaches an OD600 of 0.375 (do not allow it to go beyond as it will affect their competence).

4. Centrifuge the cells at 5000 rpm for 10 minutes at 4˚C in a JA10 rotor (pour off the supernatant immediately).

5. At 4˚C resuspend the cells in 10 mL of ice-cold solution A and repeat the 5000 rpm centrifugation for 10 minutes in a JA17 rotor (pour off the supernatant immediately).

6. At 4˚C resuspend the cells in 10 mL of ice-cold solution B and place the suspension on ice for 30 minutes in the cold room.

7. Spin down the cells at 5000 rpm for 5 minutes at 4˚C in a JA17 rotor (pour off the supernatant immediately).

8. At 4˚C resuspend the cells in 1 mL of solution B and then mix the suspension with 5 mL of solution B + glycerol.

9. Store at -80˚C in aliquots of 75 uL.

Reagents: Solution A 10 mM MOPS pH 7.0. 10 mM RbCl.

Solution B 100 mM MOPS pH 7.0. 50 mM CaCl2. 10 mM RbCl.

Solution B + glycerol 100 mM MOPS pH 7.0. 50 mM CaCl2. 10 mM RbCl. 50% glycerol.

All solutions should be made fresh, filter sterilized (filter through a 0.22um filter) and be equilibrated to 4˚C before use.