Add approximately 1μg of DNA to the Agrobacterium aliquot and mix gently (or just 1-2μl of plasmid)
Incubate on ice for 30 min
Transfer the tubes to liquid nitrogen until the aliquot is solidified (approx. 15 sec)
Transfer the tubes immediately to 37°C and incubate for 5 min (no longer!)
Add 100 μl of medium (LB, no antibiotics) to the tube and incubate for 2-3 hours at 28°C
Plate all on LB agar containing the appropriate antibiotics. Always take along a negative control, especially when selecting on kanamycin (Agrobacterium can revert to Kan resistant)
Leave upside down for 36-48 hours in 28°C
Pick several colonies and grow O/N in a single 50ml falcon containing 10ml LB with appropriate antibiotics
Make 25% glycerol stocks, snap-freeze in liquid nitrogen and store in -80°C