Team:Newcastle/Labwork/4 August 2009

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Lab Session 04/08/2009

Sporulation Tuning Team

Prof. Anne Moir from Sheffield University kindly sent us the spores of the genes cwlD and sleB/cwlJ

  • HC401 (FrpC2, cwlJ:: Kan, sleB-lacZ Ery R)
  • AM1877 (cwlD,Cm r, trp C2)

In the protocol given to us by Prof. Moir, there are two methods (Methods A and B), which can be used to recover the cwlD spores. However, neither give complete restoration of germination.

[Attach Prof. Moir's protocols]

Referring to Prof. Moir's protocols, Method A is a fast method which results in partial germination of approximately 0.1%. Method B on the other hand, is a slow method which involves stripping of the spore coat layers for improved germination of approximately 10% recovery. Due to the lack of time, the group decided to go with Method A.

Preparation

Items needed
  • Lysozyme (not available, ordered)
  • 10mM (0.01M) Potassium phosphate (Crystal form available, ordered)
  • 50mM (0.05M) KCl (Stock solution of 1M available)
  • 1mM (0.001M) MgCl2 (Stock solution of 0.5M available)
  • L-alanine (not available, ordered)


0.01M Potassium Phosphate Solution

MW * Desired Volume (L) * Desired Molarity (M) 174.18 * 0.1L * 0.01M = 0.174g

Therefore, 0.174g of Potassium Phosphate is mixed with 0.1L of DI water to make 0.1L of 0.01M Potassium Phosphate


0.05M KCl Solution

1M (Stock Solution) / 0.05M (Desired Molarity) = 20 Therefore, in order to obtain a solution of 0.05M, the stock solution needs to be diluted 20 times.

Desired volume = 0.1L 100ml/20=5ml of KCl, and DI water would make up 95ml, producing 100ml of 0.05M KCl.






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