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Team:Newcastle/Transformation of B. subtilis
From 2010.igem.org
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Contents |
Transformation of Bacillus subtilis
Materials required
- Bacillus subtilis 168
- 10 µl of DNA
- SMM medium (1 liter)
- 2.0 g of ammonium sulphate
- 14.0g of dipotassium hydrogen phosphate
- 6.0g of potassium dihydrogen phosphate
- 1.0g of sodium citrate dehydrate
- 0.2g of magnesium sulphate
- Top up the rest of the medium with water
- MM competence medium
- 10 ml of SMM medium
- 125 µl of Solution E (40% glucose)
- 100 µl of Tryptophane solution (at a concentration of 2mg/ml) –
- 60 µl of Solution F (1M MgSO4)
- 10 µl of 20% Casamino acids
- 5 µl of 0.22% Fe-NH4-citrate
- Starvation medium
- 10 ml of SMM medium
- 125 µl of Solution E (40% glucose)
- 60 µl of Solution F (1M MgSO4)
Protocol
This protocol will stretch for 2 days.
Protocol for Day 1
- Inoculate a single colony of Bacillus subtilis 168 into a 15 ml falcon tube containing 10 ml of MM competence media.
- For control transfer 10 ml of MM competence media without the bacteria.
- Incubate overnight in a shaking incubator at 37ºC.
Protocol for Day 2
- Warm up the starvation medium to 37ºC.
- Transfer 0.6 ml of the overnight culture into 50 ml falcon tube containing 10 ml of MM competence medium.
- Incubate the tubes for 3 hours at 37ºC.
- Add 10 ml of starvation medium (prewarmed) into each tube and incubate for a further 1-2 hours at 37ºC.
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