Jonesma Lab:Protocols:tobaccotransform

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Transforming Tobacco

  1. O/N starter culture (10ml with appropriate antibiotics; GV3101 should be grown in Rif, Gent and plasmid-specific Ab), at 28°C
  2. Spin down 2 ml of O/N culture at 3000 rpm for 15 min
  3. Resuspend in 1 mL Infiltration Buffer
  4. Spin down at 3000 rpm for 15 min
  5. Measure OD
  6. Mix cultures together if needed, bringing to a final OD of 0.1 per culture
  7. Add acetosynringone (1/1000 dilution of stock)
  8. Leave shaking at RT for 2+ hours
  9. Infiltrate using 1 mL syringe
  10. Leave tobacco on bench/dark growth room overnight
  11. Spray with luciferin (1x)
  12. Move to 30 μE red light for 24 hrs
  13. Spray with luciferin (1x)
  14. Image (Typically 3 x 2hr red, 6 x 2hr condition of interest, 3 x 2hr red

Infiltration Buffer

10 mM MES

10 mM MgCl2

Make up with dH2O

pH5.7 (KOH)

Acetosynringone stock

150 mg/mL dissolved in DMF