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