Transformation of Bacterial Cultures Using Hexamine Cobalt Chloride
Prepare the SOB medium .
Prepare the SOC medium by using the SOB media supplemented with 20mM glucose .
Prepare the TFB buffer .
Prepare the DTT solution .
Grow 5 mL of the host cells overnight in SOB media at 37°C .
Inoculate 40 mL of SOB medium with 0.8 mL of the overnight culture .
Grow to an A550 of 0.45 - 0.55 at 37°C ( approximately 3 - 4 hours ) .
Centrifuge the cells in the Sorvall SS34 rotor at 5,000 rpm , 5 min , 4°C .
Discard the supernatant .
Resuspend the pellet with 12.5 mL of the TFB solution .
Hold the remaining 2.5 mL of TFB for use later .
Chill the cells on ice for 15 min .
Centrifuge the cells in the Sorvall SS34 rotor at 5,000 rpm , 5 min , 4°C .
Discard the supernatant .
Resuspend the pellet with 2.4 mL of TFB solution .
Add DMSO to 3.5 % ( 84µL ) , mix and chill on ice for 5 min .
Add DTT solution to 75 mM ( 84 µL ) , mix and chill on ice for 10 min .
Add an equal volume of DMSO as before ( 84 µL ) , mix and chill on ice for 5 min .
The cells are now " competent " .
Pipet 21 µL competent cells per prechilled microfuge tube .
Add the DNA ( in as small a volume as possible , 1 - 2 µL / tube ) , mix and chill on ice for 30 min .
Heat pulse the tubes at 42°C for 3 min .
Add 80 µL of SOC medium per tube and incubate the tubes at 37°C for 60 min .
Spread 100 µL onto each plate .
Incubate the plates at 37°C overnight .
Then chill on ice for 2 min .
