Arabidopsis Transformation Procedure Protocol (June Medford’s Lab)
- Grow a starter culture of Agrobacterium (2mL) containing the desired binary plasmid on LB media supplemented with the appropriate antibiotics (20mg/L Gentamicin for the Agrobact.), at 30oC / 2days.
- Inoculate this starter culture into 250 mL of LB media (containing the same antibiotics) contained in 100mL flasks and grow them at 30oC for about 1 day (according to Andrew Bent the O.D. of the bacteria does not influence the efficiency of transformation.)
- Transfer the media to centrifuge bottles, and spin the cells at 6000 rpm for 12 minutes at 4oC.
- Discard the supernatant and resuspend the bacterial cells in 500 mL of ‘New’ Infiltration Media containing 25 mL 0.005% Silwet L-77 (Lehle Seeds).
- Transfer the bacteria-containing infiltration media to a 4 L plastic beaker.
- Invert the pots containing the Arabidopsis plants, and dip them in the solution for about 1 minute, making sure the flower buds are immersed in the solution (it is not necessary to dip the leaves, but it is ok if they are).
- After dipping the plants, transfer them to a flat, with the pots lying on their sides.
- Cover the flat with Saran Wrap and take it back to the growth chamber at 25oC.
- After ON, remove the Saran Wrap and place the plants upright.
10. Water the plants regularly until the siliques are formed. Siliques first turn yellow, then turn brown as they ripen.
11. Harvest Seeds.
12. Select for transformants according to resistance conferred by the plasmid.