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 .
