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Arabidopsis floral dip transformation

1) Start Arabidopsis seeds 2-3 weeks before you plan on doing the transformation. I have found that you will get the healthiest plants when the light intensity is not too high. In our growth rooms, that means turn off the incandescent lights and one set of the fluorescent lights. It also seems that the plants grow really well with 14 hour days, but 16 hours is OK too. Plants are ready when flowers are starting to open, but before siliques are formed.

2) Once you have a construct in Agrobacterium, be sure to streak a single colony every couple of days to make the initial culture grow faster.

3) In the morning, pick one colony from a freshly streaked plate and inoculate a 5 ml culture in the morning containing LB+ kan50+gent100 and grow at 28 C.

4) In the evening, inoculate a 500 ml culture with the same antibiotics with 5 ml of the initial culture. If the initial culture is cloudy, add less to the the 500 ml culture.

Alternatively, grow 10 ml initial culture overnight and inoculate the 500 ml culture in the morning. It usually takes a long time to get the 500 ml culture to the proper OD, so I prefer the first method.

You will have to optimize the proper amount of time and size of your cultures to meet your time constraints. I find that the OD600 of the 500 ml agrobacterium culture doubles roughly every 90 minutes (once it is growing exponentially).

5) Grow the 500 ml culture until the OD600 reaches 0.6-0.8.

6) Collect cells by centrifuging for 15 min in swing bucket (divide culture into 50 ml falcon tubes).

7) Discard supernatant and resuspend cells in 300 ml of 5% sucrose, 0.1 % Silwet. (per 300 ml: 15 g sucrose, 30 ul Silwet).

8) Swish plants in resuspended cell solution until flowers have a slight film on them (~20 sec).

9) Lay plants on side in plastic tray with plastic dome over top. Next day, move the dome partially off in morning. In the evening, remove dome, stand plants up, and place in growth room.

10) Repeat dipping procedure one to 2 more times every 7 days.

11) Water plants normally and collect seeds when dry for testing.

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