Transformation of Bacillus subtilis

Materials required

1. Bacillus subtilis 168
2. 10 µl of DNA
3. 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
4. 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
5. 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

1. Inoculate a single colony of Bacillus subtilis 168 into a 15 ml falcon tube containing 10 ml of MM competence media.
2. For control transfer 10 ml of MM competence media without the bacteria.
3. Incubate overnight in a shaking incubator at 37ºC.

Protocol for Day 2

1. Warm up the starvation medium to 37ºC.
2. Transfer 0.6 ml of the overnight culture into 50 ml falcon tube containing 10 ml of MM competence medium.
3. Incubate the tubes for 3 hours at 37ºC.
4. Add 10 ml of starvation medium (prewarmed) into each tube and incubate for a further 1-2 hours at 37ºC.

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