Chemical comp. E.coli

14/07/10

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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.

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Dette sted blev sidst opdateret 14. July 2010