Agroinfiltration methods (FWD)

1, Use the stab culture supplied as a starting material and inoculate 5 mI overnight culture
(28'C for agro):
-5 ml L-broth
-5 µI tetracycline (5 µl/ml final) (Depend on the constructs)
-5 µI kanamycin (50 µl/ml final)  (Depend on the constructs)

2, Use all the 1 ml of the overnight culture to inoculate 50 ml L-broth and grow overnight:
-50 ml L-broth
-50 µI tetracycline (5 µglml final)
-50 µI kanamycin (50 µglml final)
-500µI MES (10 mM final) [optional]
-I0 µI Acetosyringone (20 µM final)

3, Precipitate the bacteria and resuspend the pellet in solution containing:
-10 mM MgCI2
-10 mM MES
-100 µM Acetosyringone

4, Leave on the bench for 2 to 3 hours before agroinfiltration (or overnight).

5, Perform the infiltration with a 2ml syringe. Simply press the syringe (no needle) on the
underside of the leaf, and exert a counter-pressure with your finger on the other side.
Infiltrate the maximum number of leaves, avoid cotyledons.


From David Baulcombe's website