- Make Resuspension solution: 10% glycerol, 20 mM CaCl2, autoclaved. Keep at 4°C before use.
- Pick a single colony of the desired Agrobacterium strain from a plate and inoculate in 10 ml of modified YEB (or LB) with the appropriate antibiotics
- Incubate at 28°C, 180 rpm O/N
- Transfer 1ml of pre-culture to 100 ml of YEB (or LB) with the appropriate antibiotics in a 500 ml Erlenmeyer and grow O/N 28°C, 180 rpm
- Incubate the culture on ice for 15 minutes
- Centrifuge the culture in falcon tubes at 2000xg for 15 min at 4°C. Note: take ~25ml per 50ml falcon tube to make sure the cells pellet well
- Discard the supernatant making sure all medium is gone
- Resuspend cells delicately in 1 ml of pre-chilled resuspension solution, keeping cells in ice the whole time
- Once the pellet is entirely resuspended, aliquot in pre-chilled eppendorf tubes, 50 μl per tube and freeze immediately in liquid nitrogen
- Store at -80°C.
100ml of culture yields 40 competent Agrobacterium shots (resuspend in 2ml resuspension solution)