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transient transformation and expression

note: we use this protocol for transient transformation of Nicotiana benthamiana leaves

  1. streak out Agrobacterium from –80°C and let grow over night on selective medium @ 30°C
  2. grow 2 ml over night culture in selective medium in glass tubes @ 30°C
  3. spin down in 2 ml tubes (4 min; 10000 rpm)
  4. remove SN
  5. resuspend in infiltration medium (appr. ml)
  6. dilute to an OD of 1.0 (for protein expression (WB, microscopy…)
  7. mix with P19 strain (silencing inhibitor strain) 1:1 → incubate for 1 h or more
  8. infiltrate leaves (normally lower side) with a syringe, label infiltrated area with edding
  9. (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)