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Team:TU Darmstadt/Notebook/Methods/Chemically competent cells
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Revision as of 16:18, 16 October 2014
Chemically competent cells
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
- Glycerine
Procedure:
The transformation of E. coli with plasmid DNA via heatshock transformation requires chemically competent cells.
Inoculate 2 mL of LB-Media with an E. coli colony and incubate at 37 °C overnight.
Inoculate 200 mL LB with the preculture.
Incubate at 37°C and 150 rpm until an OD600 of 0.4-0.6 is reached.
Incubate cells on ice for 15 min.
Centrifuge the culture at 4°C and 3000 x g for 10 min (the following steps are carried out on ice).
Resuspend cell pellet in 10 mL ice cold 100 mM CaCl2 (Do not vortex!).
Incubate on ice for 1 hour.
Centrifuge the culture at 4°C and 3000 x g for 10 min.
Resuspend cell pellet in 10 mL ice cold 100 mM CaCl2.
Incubate on ice for 1 hour.
Centrifuge the culture at 4°C and 3000 x g for 5 min.
Resuspend cell pellet in 2mL ice cold 100 mM CaCl2 and 15 % (v/v) glycerine.
Incubate on ice for 30 min.
Aliquot the cells à 100 µL.
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 glycerine
- Add ddH2O to 50 ml
- Sterilize by autoclaving
References:
Mandel, M. and Higa, A.: Calcium-dependent bacteriophage DNA infection. J Mol Biol, 1970, 53, 159-162
