Team:TU Darmstadt/Protocols/Chemically competent cells

From 2012.igem.org

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. 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 100µL
  15. Store at -80°C

Solutions

  • CaCl2
    • 5.55 g CaCl2
    • Add di H2O to 1 L
    • Sterilize by autoclaving
  • Cryo solution
    • 0.278 g CaCl2
    • 10 ml glycerin
    • Add di H2O to 50 ml
    • Sterilize by autoclave

References

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