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Preparing competent Agrobacterium cells
- 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.
- 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).
- Add culture to 50 ml falcon tube, chill on ice, and centrifuge for 20 minutes at 4C in swing bucket centrifuge (max speed).
- 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.
- Freeze in liquid nitrogen and store at -80.