Jonesma Lab:Protocols:tobaccotransform
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Transforming Tobacco
- O/N starter culture (10ml with appropriate antibiotics; GV3101 should be grown in Rif, Gent and plasmid-specific Ab), at 28°C
- Spin down 2 ml of O/N culture at 3000 rpm for 15 min
- Resuspend in 1 mL Infiltration Buffer
- Spin down at 3000 rpm for 15 min
- Measure OD
- Mix cultures together if needed, bringing to a final OD of 0.1 per culture
- Add acetosynringone (1/1000 dilution of stock)
- Leave shaking at RT for 2+ hours
- Infiltrate using 1 mL syringe
- Leave tobacco on bench/dark growth room overnight
- Spray with luciferin (1x)
- Move to 30 μE red light for 24 hrs
- Spray with luciferin (1x)
- 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