Cloning

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.

Make Resuspension solution: 10% glycerol, 20 mM CaCl2, autoclaved. Keep at 4°C before use. Pick a single colony of the desired Agrobacterium strain from a plate and inoculate in 10 ml of modified YEB (or LB) with the appropriate antibiotics Incubate at 28°C, 180 rpm O/N Transfer 1ml of pre-culture to 100 ml of YEB (or LB) with the appropriate antibiotics in a 500 ml Erlenmeyer and grow O/N 28°C, 180 rpm Incubate the culture on ice for 15 minutes Centrifuge the culture in falcon tubes at 2000xg for 15 min at 4°C.

Grow 5 ml ON culture, 37°C, 180 rpm with appropriate antibiotics Inoculate 100 ml LB with antibiotics with ON culture 1:100 Shake at 37°C until culture reaches OD600 ~0.5 (usually ~3h), put buffers on ice Spin cells at 2000 x g, 4°C, 5 min using two Falcon tubes and resuspend each pellet in 10 ml ice cold TFB I After a short incubation on ice spin cells down at 2000 x g, $°C, 5 min Resuspend each pellet in 2 ml ice cold TFB II and incubate on ice for 30 min.

Day 1

Thaw competent cells on ice ~5min Add 50μl of competent cells to 1μl plasmid or 5μl of ligation mix (no more than 10% ligation mix final volume) in a PCR tube Incubate on ice 30min Heat-shock in PCR machine 42°C 45s (no heated lid) Place 2min on ice Add 100μl LB Recover 45-60min 37°C. When using antibiotics which do not target the ribosomes (e.g. Ampicillin etc.) this step can be skipped Plate on plates containing appropriate antibiotics.