Jonesma Lab:Protocols:tobaccotransform

Transforming Tobacco

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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