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note: we use this protocol for transient transformation of Nicotiana benthamiana leaves
- streak out Agrobacterium from –80°C and let grow over night on selective medium @ 30°C
- grow 2 ml over night culture in selective medium in glass tubes @ 30°C
- spin down in 2 ml tubes (4 min; 10000 rpm)
- remove SN
- resuspend in infiltration medium (appr. ml)
- dilute to an OD of 1.0 (for protein expression (WB, microscopy…)
- mix with P19 strain (silencing inhibitor strain) 1:1 → incubate for 1 h or more
- infiltrate leaves (normally lower side) with a syringe, label infiltrated area with edding
- (24) – 48 (better) hpi: collect leaf material with Korkbohrer
infiltration medium:
- 10 mM MES pH5.3 – 5.5 (stock 1 M → for 100 ml = 1 ml)
- 10 mM MgCl2 (stock 1 M → for 100 ml = 1 ml)
- 150 µg/ml Acetosyringone (in DMF) (stock 150 mg/ml → for 100 ml = 100 µl)
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