Direct Agrobacterium transformation of GV3101(pMP90RK) with pLH vectors

Direct Agrobacterium transformation of GV3101(pMP90RK) with pLH vectors

Although the transformation frequency by this method is low compared to the triparental mating method, the technique is reliable and very rapid. This transformation procedure also eliminates much of the plasmid rearrangement that often occurs during triparental mating.

A. 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.

 

B. 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.


C. Antibiotic selection

  • Agrobacterium (GV3101)

Rifampicin 100 µg/ml

   
  • Ti-plasmid (pMP90RK)

Gentamycin 50 µg/ml,
Kanamycin 50 µg/ml

   
  • Binary (pLH vectors)

Spectinomycin 100 µg/ml,
Streptomycin 300 µg/ml