Preparation of of Agrobacterium competent cells and direct transformation by freeze-thaw method

• Streak Agrobacterium strain on YEM plate containing rifampicin (stock 20 mg/ml; dissolve in methanol). Grow at 28 C for 48 hrs.

• Composition of YEM medium (1 Lt)

  • Mannitol 10g

  • Yeast extract 0.4g

  • NaCl 0.1g

  • MgSO4, 7H2O 0.02g

  • 0.5% K2HPO4 10ml (separately prepare with autoclaved water, filter sterilize and add at the time of pouring plate)

  • Agar 15g

• Inoculate single colony in 5 ml YEM + Rif. And grow at 28 C for 12-16 hours.

• Inoculate 2 ml culture to 50 ml YEM + Rif in a 250 ml flask and shake vigorously (250 rpm) at 28 C until culture grows to an OD600 of 0.5-1.0 (5-6 hrs)

• Chill the culture on ice. Centrifuge cell suspension at 3000 xg for 5 min at 4 C.

• Resuspend cells in 1 ml of 20 mM CaCl2 (0.03 g CaCl2 in 10 ml autoclaved water, filter). Centrifuge at 300 xg for 5 min at 4 C. Discard supernatant. Resuspend pellet in 1 ml 20 mM CaCl2. Dispense 0.1 ml of aliquots into prechilled EP tubes. Freeze in liquid nitrogen and store at -80 C.

Transformation (freeze-thaw method)

• Add about ~1 m g of plasmid DNA into the 100 m l of competent cell. Freeze the cells by placing in liquid nitrogen.

• Immediately thaw cells by transferring to 37 C for 5 min.

• Add 1 ml of YEM to the cells and incubate at 28 C for 4 hrs.

• Spread the cells (200 m l/plate) on YEM agar with appropriate antibiotic.

• Storing of bacterial cultures for long term storage: 800 m l culture + 200 m l of autoclaved glycerol (absolute).