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. 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