Team:TU Darmstadt/protocols/Chemically competent cells

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Revision as of 00:04, 5 October 2013







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
  • - Glycerin


Procedure


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

  1. Inoculate 2 mL of LB-Media with an E. coli colony and incubate at 37 °C overnight.
  2. Inoculate 200 mL LB with the preculture.
  3. Incubate at 37°C and 150 rpm until an OD600 of 0.4-0.6 is reached.
  4. Incubate cells on ice for 15 min.
  5. Centrifuge the culture at 4°C and 3000 x g for 10 min (the following steps are carried out on ice).
  6. Resuspend cell pellet in 10mL ice cold 100 mM CaCl2 (Do not vortex!).
  7. Incubate on ice for 1 hour.
  8. Centrifuge the culture at 4°C and 3000 x g for 10 min.
  9. Resuspend cell pellet in 10mL ice cold 100 mM CaCl2.
  10. Incubate on ice for 1 hour.
  11. Centrifuge the culture at 4°C and 3000 x g for 5 min.
  12. Resuspend cell pellet in 2mL ice cold 100 mM CaCl2 and 15 % (v/v) glycerine.
  13. Incubate on ice for 30 min.
  14. Aliquot the cells à 100µ.
  15. Store at -80°C.


Mixtures


CaCl2-Solution

  • - 5.55 g CaCl2
  • - add ddH2O to 1 L
  • - sterilize by autoclaving


Cryo solution

  • - 0.278 g CaCl2
  • - 10 ml glycerin
  • - Add ddH2O to 50 ml
  • - Sterilize by autoclave

References

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

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