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.
