Team:TU Darmstadt/Protocols/Chemically competent cells

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