Last Update: December 2006
1. LB media (1 L):
Mix 10 g of Bacto-tryptone, 5 g of yeast extract, and 10 g of NaCl. pH to 7.5 with NaOH and add dH2O to 1 L (Autoclave)
1. For Agrobacterium tumefaciens strain GV3001 streak on LB plate + 50 mg/mL rifampicillin, and 50 mg/mL gentamicin (for strain LBA4404: LB plate + 50 mg/mL streptomycin)
2. Streak Agrobacterium tumefaciens strain on LB plate + selection
Allow to grow at 28oC (1-3 days). Agrobacterium-harboring Ti plasmids are cultured at a lower temperature to prevent "curing" of the Ti-plasmid, therefore growth is slower

NOTE: do not grow agrobacterium on YEP media when making electrocompetent cells

3. Place several colonies in 100 mL LB media containing selection, grow over night at 28oC
4. Transfer 50 mL cell culture into fresh 500 LB media in 2.5 L flask. Allow cell to grow at 28oC (200 rpm), until OD600 = 1-1.5 (~4 h)
5. Transfer cell culture to 2 cold centrifuge bottles (250 mL), and place tubes on ice for 20 min
6. Centrifuge cells in Sorval GSA rotor at 4oC for 15 min at 4,000 xg

7. Pour off media completely and resuspend cells in each tube completely but gently with 250 mL of cold sterile water

8. Centrifuge cells in Sorval GSA rotor at 4oC for 15 min at 4,000 xg
9. Repeat steps 7-8 four more times
NOTE: it is very important to wash cells with excess water in order to remove salts

10. Pour off media and resuspend cells in each tube with 25 mL of cold sterile 10% glycerol. Combine the suspended cells from each tube into one cold 50 mL polypropylene Falcon centrfuge tube

11. Centrifuge cells using Sorval RT6000B rotor at 4oC for 10 min at 3,000 g (3500 rpm = #8)

12. Pour off media and finally resuspend cells completely but very gently in 2 mL of cold 10% glycerol (*Use glycerol from autoclaved 80% glycerol stock)

13. Divide into 80 mL aliquots (60 tubes) and freeze the cells in liquid nitrogen. Cells can be used immediately or stored at -80oC and can be used for transformation for at least 6 months