< IGEM:Harvard | 2010 | General Protocols
- Grow healthy Arabidopsis plants until they are flowering. Grow under log days in pots in soil covered with bridal veil, window screen or cheesecloth.
- (Optional) Clip first botls to encourage proliferation of many secondary bolts. Plants will be ready 406 days after clipping. Clipping can be repeated to delay plants. Optimal plants have many immature flower clusters.
- Grow 3mL O/N cultures of construct in AgroGV3101 with proper antibiotics selection
- Inoculate into 250mL LB kan/gent (does 4-6 pots)
- Spin down, 7k X 5min
- Resuspendto OD600=0.8 (can be higher or lower) in 5% sucrose, 0.05% Silwet L-77 (50g sucrose, 0.5mL Silwet, 1L distilled water). If there are problems with toxicity, use 0.02% or as low as 0.005%. You will need 100-200mL for each two or three small pots to be dipped, or 400-500 mL for each 2-3 3.5" (9cm) pots.
- Dip above ground parts of plant in solution for 2-3 seconds, with gentle agitation. You should then see a film of liquid coating plant.
- Place dipped plants under a dome or cover for 16-24h to maintain high humidity (plants can be laid on their side if necessary). Do not expose to excessive sunlight.
- Water and grow plants normally, tying up loose bolts by some means. Stop watering as seeds become mature.
- Harvest dry seed. Tranformatns are usually all independent, but are guarantted to be independent if they come off of separate plants.
- Select for transformants using antibiotic or herbicide selectable marker on agar plates. For example, vapor-phase sterilize and plate 40mg = 2000 seed (resuspended in 4mL 0.1% agarose) on .5X MS/0.8% tissue culture Agar plates with antibiotic, cold treat for 2 days, and grow under continuous light for 7-10 days.
- Transplant putative transformants to soil. Grow, test and use.
For higher rates of transformation, plants may be dipped 2-3 times at 7 day intervals. It is suggested to do one dip 2 days after clipping, and a second dip one week later. Do not dip less than 6 days apart.