Start by diluting 0.5 microliters of the overnight cultures of Agrobacterium tumefaciens culture with 50 milliliters of autoclaved ultrapure water. Spread 10 microliters of this diluted culture on a lysogeny broth or LB plate. Incubate the plate at 28 to 30 degrees Celsius for one to three days.
Now, inoculate a single colony in 20 milliliters of LB media supplemented with rifampicin. Incubate the culture overnight at 28 to 30 degrees Celsius. The next day, inoculate 500 milliliters of plain LB media in a shaker flask with nine milliliters of overnight culture.
Place the cultures on a shaker at 28 to 30 degrees Celsius. Following incubation and dividing the cultures into tubes, centrifuge the chilled tubes at four degrees Celsius and 4, 000 G for 15 minutes. Using a pipette, remove the supernatant and resuspend each pellet in 50 milliliters of ice cold water.
After centrifuging and discarding the supernatant, resuspend the pellet in 25 milliliters of ice cold water in each tube. Centrifuge as before, then resuspend the pellet in one milliliter of ice cold 10%glycerol. Now, combine the contents of all the tubes into a single 50-milliliter tube before centrifuging.
Finally, after discarding the supernatant, resuspend the pellet in 400 microliters of ice cold glycerol.
