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.
- 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 10mL 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 10mL 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 100µ.
- 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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