Team:Newcastle/Transformation of B. subtilis

From 2010.igem.org

(Difference between revisions)
(Protocol for Day 2)
(Protocol for Day 2)
 
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# Eppendorf tubes
# Eppendorf tubes
# Agar plates containing the appropriate antibiotic
# Agar plates containing the appropriate antibiotic
 +
# Water bath
 +
# Centrifuge
# SMM medium (1 liter)
# SMM medium (1 liter)
#* 2.0 g of ammonium sulphate
#* 2.0 g of ammonium sulphate
-
#* 14.0g of dipotassium hydrogen phosphate
+
#* 14.0 g of dipotassium hydrogen phosphate
-
#* 6.0g of potassium dihydrogen phosphate
+
#* 6.0 g of potassium dihydrogen phosphate
-
#* 1.0g of sodium citrate dehydrate
+
#* 1.0 g of sodium citrate dehydrate
-
#* 0.2g of magnesium sulphate
+
#* 0.2 g of magnesium sulphate
#* Top up the rest of the medium with water  
#* Top up the rest of the medium with water  
# MM competence medium
# MM competence medium
#* 10 ml of SMM medium
#* 10 ml of SMM medium
#* 125 µl of Solution E (40% glucose)
#* 125 µl of Solution E (40% glucose)
-
#* 100 µl of Tryptophane solution (at a concentration of 2mg/ml) –
+
#* 100 µl of Tryptophane solution (at a concentration of 2 mg/ml) –
#* 60 µl of Solution F (1M MgSO4)
#* 60 µl of Solution F (1M MgSO4)
#* 10 µl of 20% Casamino acids
#* 10 µl of 20% Casamino acids
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==Protocol==
==Protocol==
-
This protocol will stretch for 2 days.
+
This protocol will stretch for 2 days and aseptic technique have to be applied for all steps.
===Protocol for Day 1===
===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.
# 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.
+
# For control, transfer 10 ml of MM competence media without the bacteria.
# Incubate overnight in a shaking incubator at 37ºC.
# Incubate overnight in a shaking incubator at 37ºC.
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# Warm up the starvation medium to 37ºC in a water bath.
# Warm up the starvation medium to 37ºC in a water bath.
# Add 10 ml of starvation medium (prewarmed) into each tube and incubate for a further 1-2 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.
+
# Transfer 0.4 ml of the medium not containing ''B. subtilis'' into one Eppendorf tube.
#* Add 10 µl of water into the tube.
#* Add 10 µl of water into the tube.
-
# Transfer 2X 0.4 ml of the medium containing ''B. subtilis'' into two Eppendorf tubes.
+
# Transfer 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 DNA into one tube.
#* Add 10 µl of water into one tube.
#* Add 10 µl of water into one tube.
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# Resuspend the pellet in the remaining supernatant and plate it onto agar plates containing the appropriate antibiotic.
# Resuspend the pellet in the remaining supernatant and plate it onto agar plates containing the appropriate antibiotic.
# Incubate the plates overnight at 37ºC.
# Incubate the plates overnight at 37ºC.
 +
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Latest revision as of 14:22, 26 October 2010

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Contents

Transformation of Bacillus subtilis

Materials required

  1. Bacillus subtilis 168
  2. 10 µl of transformation DNA
  3. Pipettes
  4. 2X 15 ml falcon tubes
  5. 2X 50 ml falcon tubes
  6. Eppendorf tubes
  7. Agar plates containing the appropriate antibiotic
  8. Water bath
  9. Centrifuge
  10. SMM medium (1 liter)
    • 2.0 g of ammonium sulphate
    • 14.0 g of dipotassium hydrogen phosphate
    • 6.0 g of potassium dihydrogen phosphate
    • 1.0 g of sodium citrate dehydrate
    • 0.2 g of magnesium sulphate
    • Top up the rest of the medium with water
  11. MM competence medium
    • 10 ml of SMM medium
    • 125 µl of Solution E (40% glucose)
    • 100 µl of Tryptophane solution (at a concentration of 2 mg/ml) –
    • 60 µl of Solution F (1M MgSO4)
    • 10 µl of 20% Casamino acids
    • 5 µl of 0.22% Fe-NH4-citrate
  12. 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 and aseptic technique have to be applied for all steps.

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. Transfer 0.6 ml of the overnight culture into 50 ml falcon tube containing 10 ml of MM competence medium.
  2. Incubate the tubes for 3 hours at 37ºC.
  3. Warm up the starvation medium to 37ºC in a water bath.
  4. Add 10 ml of starvation medium (prewarmed) into each tube and incubate for a further 1-2 hours at 37ºC.
  5. Transfer 0.4 ml of the medium not containing B. subtilis into one Eppendorf tube.
    • Add 10 µl of water into the tube.
  6. Transfer 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.
  7. 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.
  8. Centrifuge the Eppendorf tubes at 13,000 rpm for 2 minutes.
  9. Discard 0.3 ml of supernatant from each Eppendorf tubes.
  10. Resuspend the pellet in the remaining supernatant and plate it onto agar plates containing the appropriate antibiotic.
  11. Incubate the plates overnight at 37ºC.


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