Transformation of Bacterial Cultures Using The Calcium Chloride Procedure
Grow 5 mL of the host cells overnight in L broth at 37°C.
Inoculate 100 mL of L broth with 1 mL of the overnight culture.
Grow the cells with shaking at 37°C to a density of approximately 5 X 107 cells/ml.
Chill the culture on ice for 10 min.
Centrifuge the cell suspension in the Sorvall SS34 rotor at 4,000g, 5 min, 4°C.
Discard the supernatant.
Resuspend the cells with 1/2 the original volume with an ice cold, sterile solution of 10 mM Tris-HCl, pH 8.0, 50 mM CaCl2.
Place the cell suspension in an ice bath for 15 min.
Discard the supernatant.
Resuspend the cells with 1/15 of the original volume with an ice cold, sterile solution of 10 mM Tris-HCl, pH 8.0, 50 mM CaCl2.
Dispense 0.2 mL aliquots into prechilled microfuge tubes.
Store the cells at 4°C for 12-24 hours.
Add the DNA in as small a volume as possible (1-2 µL/plate).
Mix and store on ice for 30 min.
Transfer the mixtures to 42°C for 2 min.
Add 1.0 mL of L broth to each tube and incubate at 37°C for 30 min (tetracycline selection) or 60 min (ampicillin or kanamycin selection) without shaking.
Spread an appropriate volume (usually 100-200 µL) of cells onto selective media by using either the spread plate method or the agar overlay method.
Leave the plates at room temperature until the agar overlay has solidified or the liquid has been absorbed into the plate.
Invert the plates and incubate at 37°C.
Colonies should appear in 12-16 hours.
Centrifuge the suspension in the Sorvall SS34 rotor at 4,000g, 5 min, 4°C.
