Revision as of 15:49, 5 August 2010

Transformation of Bacillus subtilis

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)

This protocol will stretch for 2 days.

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.

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.
Transfer 1X 0.4 ml of the medium not containing B. subtilis into one Eppendorf tube.
- Add 10 µl of water into the tube.
Transfer 2X 0.4 ml of the medium containing B. subtilis into two Eppendorf tubes.
- Add 10 µl of DNA into one tube.
- Add 10 µl of water into one tube.
Incubate the samples for 1 hour at 37ºC in the shaking incubator.
- The Eppendorf tubes have to be aerated, therefore incubate the tubes on their side.
Centrifuge the Eppendorf tubes at 13,000 rpm for 2 minutes.
Discard 0.3 ml of supernatant from each Eppendorf tubes.
Resuspend the pellet in the remaining supernatant and plate it onto agar plates containing the appropriate antibiotic.
Incubate the plates overnight at 37ºC.

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@@ Line 43: / Line 43: @@
 #* Add 10 µl of DNA into one tube.
 #* Add 10 µl of water into one tube.
+# Incubate the samples for 1 hour at 37ºC in the shaking incubator.
+#* The Eppendorf tubes have to be aerated, therefore incubate the tubes on their side.
-Transfer 0.4ml of the competence solution (WITHOUT B. subtilis) into one Eppendorf tube and transfer 0.4ml of competence solution (WITH B. subtilis)twice into two Eppendorf tubes (so that each tube contains 0.4ml). To one tube containing Bacillus subtilis add 10µl of the DNA. To the other two tubes (one containing B. subtilis and the other containing just competence solution with no B. subtilis) add 5-10µl of water. Place these in the shaking incubator for 1 hour at 37ºC. To find out how to carry out the incubation step successfully, see the ‘Important Notes’ section below.
+# Centrifuge the Eppendorf tubes at 13,000 rpm for 2 minutes.
+# Discard 0.3 ml of supernatant from each Eppendorf tubes.
+# Resuspend the pellet in the remaining supernatant and plate it onto agar plates containing the appropriate antibiotic.
+# Incubate the plates overnight at 37ºC.
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