Growth and storage of Agrobacterium tumefaciens

Strain GV3101: resistant to gentamycin and rifampicin so add 25-50 ug/ml Gentamycin, 10 ug/ ml rifampicin on plates or in liquid media for selection. GV3101 is sensitive to kanamycin (or chloramphenicol), so is a good strain for use with binary vectors that confer kan resistance (or chloramphenicol resistance) in bacteria (e.g. transformed cells will be Gent, rif and Kan resistant). GV3101 carries a disarmed Ti plasmid that possesses the vir genes needed for T-DNA transfer, but has no functional T-DNA region of its own. Grow at 28-30℃. Store as glycerol stock (800 ml of fresh overnight culture + 200 μl sterile 80% glycerol) at -80℃.

Transformation of A. tumefaciens with plasmid DNA (binary vector system)

Two methods of direct DNA transfer can be used, thus eliminating the need for the old genetic method of transferring a plasmid maintained in E. coli by triparental mating (E. coli + RK2013 + A.tumefaciens).

Method 1: Freeze/thaw shock transformation. This is not very efficient (~200 colonies per microgram of DNA), but it is really easy.

1. Pick a single colony of the Agrobacterium strain of choice and inoculate 3 ml of LB (or 2YT) in a 15 ml snap-cap tube (Falcon tube). Grow @ 30℃ overnight on a roller drum. Be sure to include the appropriate antibiotic selection (Gentamycin, rifampicin for GV3101).

2. Inoculate 50 ml of LB in a 250 ml flask with 0.5 ml (1/100 volume) of the overnight culture and grow @ 30℃ until mid-log (OD600 is between 0.5 and 1.0). In practice, I (Craig) never bother taking OD readings but grow cultures until they are dense enough to give nice, silky, cloud-like swirls of cells when the culture is held up to the light and jostled. This takes ~4-5 hours to get the cells to this stage. You could probably cut down the time by increasing the initial inoculum.

3. Chill culture 5-10 min. on ice, centrifuge @ 3000 rpm for 5 min. @ 4℃ in chilled, sterile centrifuge tubes (30 ml Corex tubes).

4. Discard supernatant, drain inverted for 30-60 seconds, and resuspend pellet in 1 ml of ice cold 20 mM CaCl2. Dispense 0.1 ml of bacterial suspension into each of two pre-chilled 1.5 ml. microfuge tubes on ice. One is a control.

5. Add 1 ug of plasmid DNA to one tube and nothing to the other the control) and mix by tapping with your index finger. Freeze tubes in liquid N2, then thaw tubes for 5 min. @ 37℃.

6. Add 1 ml of LB (or 2YT) to each tube, transfer content to a 15 ml snap-cap tube and incubate for ~2 hours on a roller drum @ 30℃.

7. Pour contets into a 1.5 ml microfuge tube and spin tubes 5 minutes at ~4K rpm to pellet cells. Remove supernatant and resuspend pellet in 100 μl of LB (or 2YT).

8. Plate all of the suspension on appropriate antibiotic-LB (or 2YT) plates and incubate for two days @ 30℃. Transformed colonies should be visible on the second day of incubation.

Method 2: Electroporation

This method is much more efficient and one can make a batch of frozen competent cells and store them at ¬80℃ for multiple experiments, which is convenient if you plan to be doing lots of cloning and transformations.

Preparation of competent cells

1. Pick a single colony of the Agrobacterium strain of choice and inoculate 3 ml of LB (or 2YT) in a 15 ml snap-cap tube (Falcon tube). Grow @ 30℃ overnight on a roller drum. Be sure to include the appropriate antibiotic selection (e.g. Gentamycin for GV3101).

2. Inoculate two 500 ml flasks each containing 100 ml of LB with 0.5 ml (1/100 volume) of the overnight culture and grow @ 30℃ with vigorous shaking until mid-log (OD600 of 0.5 - 1.0). In practice, I (Craig) never bother taking OD readings but grow cultures until they are dense enough to give nice, silky, cloud-like swirls of cells when the culture is held up to the light and jostled. It takes ~4-5 hours to get the cells to this stage. You could probably cut down the time by increasing the initial inoculum.