Transformation by the calcium chloride procedure (modified from Mandel and Higa, 1970)

Grow cell stocks on M9 minimal medium.

Day 1:
Inoculare 4 ml LB; incubate overnight

Day 2:
From the overnight culture, 2 ml is used to inoculate 100 ml of LB medium, pre-incubated at 37°C.

2 hours of vigorous shaking at 37°C (up to a cell density of approximately 10⁸ cells/ml)

Harvest cells by centrifugation: 10 minutes at 1,700.g.

Resuspend cell pellets in 50 ml ice-cold sterile CaCl₂ solution (100 mM)
Incubated on ice for 20 to 90 minutes

Concentrate cells by centrifugation

Resuspend cell pellet in 10 ml of the same CaCl₂solution.

For long-term storage, glycerol in concentrations of 10% (v/v) is added to this cell suspension, followed by rapid freezing on CO₂ ice and storage at -70°C.

Mix plasmid DNA or ligation mixture (approximately 10 ng for optimal efficiency) with 200 ml competent cells

Incubate on ice for 20 minutes

Heat-shock treatement: 5 X for 30 seconds alternately in a water bath pre-heated to 37°C and on ice.

Incubate cells on ice for 15 minutes

Add 0.8 ml of LB medium, and incubate transformation mixtures at 37°C for 30-60 min

100 ml and 900 ml fractions are spread onto selective media

Incubate the plates at 37°C for 16 hours.

Incubation of transformed cells at 30°C instead of 37°C can improve the yield of transformants (Sepulveda and Lieberman, 1999. BioTechniques 26, 1022-1024).