Transforming Arabidopsis

Ben Carter, 9/24/2014

''Note: The ideal age of plants to transform is 3-7 days after bolting. Ideal plants will have many unopened flowers and few or no mature siliques.''

Preparation of Infiltration Culture

 * 1) Streak the Agrobacterium containing the desired vector onto LB plates containing 10µg/mL rifampicin and the appropriate selective antibiotic ("media additives"). Grow in a 30oC incubator for 2 days.
 * 2) Inoculate 10mL of liquid LB medium containing the media additives with a colony from the media plate. Grow the culture overnight in a 30oC shaker-incubator.
 * 3) Confirm the identity of the vector using minipreps and diagnostic digests.
 * 4) Pour the remaining liquid culture into 500mL of sterile LB containing the media additives. Grow overnight in a 30°C shaker. 500mL is appropriate for one flat of plants. Adjust the volume to suit the number of pots being transformed.
 * 5) Centrifuge the cell culture at 6,000rpm for 10min. Decant the supernatant.
 * 6) Suspend the cell pellet in Infiltration Medium by pipetting. Use a volume of solution equal to the culture volume (i.e. 500mL for one flat).

Floral Dip

 * 1) Cover a lab cart with bench paper to avoid contamination with Agrobacterium. Perform the remaining steps on this cart.
 * 2) Prepare plastic bags with which to cover the plants. The bags should be large enough to hold 3-4 pots.
 * 3) Ensure that no pellet remains in the infiltration culture. Pour the infiltration culture solution into a small square plastic dish.
 * 4) Dip the aerial parts of the plant into the infiltration culture. Cover the soil with your hands to prevent it from contaminating the infiltration culture.
 * 5) Cover the plants in the large plastic bags 3-4 pots at a time. Fill the bag with as much air as possible by blowing into it. Twist the end of the bag closed and secure it by wrapping it with tape. If possible, prevent the plants from touching the sides of the bag.
 * 6) Allow the plants to incubate in a growth chamber overnight. Remove the bags the following day.

Infiltration Medium

 * 5% sucrose (prepare fresh)
 * 0.05% Silwet-77