Jones Lab:Protocols:Agrobacteria

Competent Agrobacteria (for cold-shock transformation)

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1. O/N starter culture (with appropriate antibiotics; GV3101 should be grown in Rif and Gent), at 28°C
2. Inoculate 500ml LB with starter culture
3. Grow at 28°C until OD 0.5-1.0
4. Chill culture on ice for 30 min
5. Spin at 10k for 15 min in SS34 at 4°C
6. Wash in 50 mL ice-cold 20mM CaCl₂
7. Spin at 10k for 15 min in SS34 at 4°C
8. Resuspend in 10 mL ice-cold 20mM CaCl₂
9. 100 μL aliquots, flash-frozen in N₂