14/07/10

Production of chemical competent E. coli cells

Perform the following steps under sterile conditions

1. Inoculate 5 ml LB and incubate over night at 37 ^oC with 200 rpm
2. Inoculate 2 x 100 ml preheated LB medium with 1 ml of the overnight culture
3. Incubate at 37 ^oC with 200 rpm until OD₆₀₀ = 0.3 – 0.4
4. Cool the cultures on ice for 15 minutes
5. Pellet cells by centrifugation 4500 rpm (Sorvall RC5) at 4 ^oC.
6. Discard the supernatant and resuspend pellet in 50 ml ice cold 0.1 M CaCl₂
7. Incubate on ice for 30 minutes
8. Pellet the cells and discard the supernatant
9. Resuspend the cells in 2 x 2.5 ml ice cold 0.1 M CaCl₂
10. Incubate for 30 minutes on ice.
11. Aliquot the resuspended cells in 100 ml portions and store at – 80 ^oC.