Team:Newcastle/Transformation of B. subtilis

From 2010.igem.org

(Difference between revisions)
(Protocol)
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==Protocol==
==Protocol==
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+
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.

Revision as of 15:27, 5 August 2010

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Contents

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.


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