- Preparation of competent cells
- Grow an Agrobacterium strain with the appropriate antibiotics in 5 ml LB medium overnight at 28°C.
- Add 2 ml of the overnight culture to 50 ml LB medium and incubate at 28°C until the culture grows to an OD600 of 0,5 to 1,0
- Chill the culture on ice. Centrifuge the cell suspension at 3000 g for 5 min at 4°C.
- Discard the supernatant solution. Resuspend the cells in 1 ml of 20 mM CaCl2 solution (ice-cold). Dispense 0,1 ml aliquots into 2 ml Eppendorf tubes.
- Freeze the cells in liquid nitrogen and store at -80°C or use directly for transformation.
- Agrobacterium transformation with binaries
- Thaw the competentcells carefully on ice
- Add 0,1-1 µg of DNA to the cells and mix.
- Freeze the cells at -80°C
- Thaw the cells by incubating in a 37°C water bath for 5 min.
- Place the cells on ice for 30 min.
- Spread the cells on prewarmed LB plates (28°C) containing all the appropriate antibiotics
- After 2 days incubation at 28°C about 20-100 colonies will appear.
- Antibiotic selection
|
Rifampicin 100 µg/ml |
|
Gentamycin 50 µg/ml, Kanamycin 50 µg/ml |
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Spectinomycin 100 µg/ml, Streptomycin 300 µg/ml |