1) Add about 1 ug of plasmid DNA to a frozen tube of competent cells (in -80; see separate protocol for preparing competent cells).
2) Thaw cells (containing plasmid DNA) at 37 C for 5 min (in a water bath).
3) Add 1 ml LB (no antibiotic) to the tube and shake gently (~ 140 RPM) for 3 hours at 28 C (this period allows the bacteria to express the antibiotic resistance gene in the plasmid).
4) Centrifuge the tubes gently for 1 minute at slow speed to pellet the cells.
5) Spread the cells on LB plate with proper antibiotics. (For GV3101, make sure to use 50 ug/ml kanamycin and 100 ug/ml gentamycin to select both for your plasmid as well as the helper plasmid - make sure your plasmid contains the gene for kanamycin bacterial resistance).
-> per 500 ml, add 500 ul of kanamycin and 1 ml gentamycin for the above concentrations.
6) Incubate at 28 C. Colonies should appear within 2-3 days.
7) Make sure to keep actively dividing Agrobacterium by streaking to a new plate every couple of days. This makes growing a large culture of agro faster for the Arabidopsis transformation.