Team:TU Darmstadt/Protocols/Chemically competent cells

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Contents

Chemically competent cells

The transformation of E. coli with plasmid DNA via heatshock transformation requires chemically competent cells.

Materials

Equipment

  • -80°C freezer
  • Incubation shaker
  • Centrifuge (cooling cababilities required!)
  • photometer
  • Ice water bath

Chemicals & consumables

  • Ice and/or liquid nitrogen
  • Falcon tubes
  • dYT Medium (50 ml p.c.)
  • ice cold 100mM CaCl2

Procedure

  1. 2mL preculture E.coli in DYT-medium
  2. incubate preculture overnight at 37°c
  3. inoculate 200 mL with the preculture
  4. incubate at 37°C and 150 rpm till an OD600 of 0.4 - 0.6 is reached
  5. incubate cells on ice for 15 min
  6. centrifuge the culture at 4°C and 3000 x g for 10 min (the following steps are done on ice)
  7. resuspend cell pellet in 10mL ice cold 100 mM CaCl2
  8. incubate on ice for 1 hour
  9. centrifuge the culture at 4°C and 3000 x g for 10 min
  10. resuspend cell pellet in 10mL ice cold 100 mM CaCl2
  11. incubate on ice for 1 hour
  12. centrifuge the culture at 4°C and 3000 x g for 5 min
  13. resuspend cell pellet in 2mL ice cold 100 mM CaCl2 and 15 % (v/v) glycerine
  14. incubate on ice for 30 min
  15. aliquot the cells à 100µL
  16. store at -80°C

References

  • Mandel, M. and Higa, A.: Calcium-dependent bacteriophage DNA infection. J Mol Biol, 1970, 53, 159-162