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Preparing competent Agrobacterium cells

  1. Grow Agrobacterium strain (we have GV3101, with its helper plasmid) in 5 mL of LB (no antibiotic) overnight at 28C. Stock culture of GV3101 is in -80 in box called vectors.
  1. Add 2 ml overnight culture to 50 ml LB in 250 ml flask and shake vigorously (250 RPM) at 28C until culture reaches OD600 of 0.5 to 1.0 (this takes several hours).
  1. Add culture to 50 ml falcon tube, chill on ice, and centrifuge for 20 minutes at 4C in swing bucket centrifuge (max speed).
  1. Discard supernatant and resuspend cells in 1 ml of 20 mM CaCl2 (ice cold). Dispense 100 ul aliquots into prechilled 1.5 ml eppendorf tubes.
  1. Freeze in liquid nitrogen and store at -80.
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