User:Stuart McKellar/Notebook/Bird Sex Testing/2012/12/03

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(Visualising lori's p2/p8 on 1% gel)
Current revision (03:53, 3 December 2012) (view source)
(Visualising lori's p2/p8 on 1% gel)
 

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Visualising lori's p2/p8 on 1% gel

Aim: differentiate between male and female loris using PCR product of p2/p8 primers and running on 1% gel.

Method: Made up a 1% gel using the life technologies SYBR safe green dye. No dye was added, as the soln was already a 1:1 dilution of dye in TBE buffer. It was easy to make up and dissolved the agar readily.

Results: Gels can be seen here: [1]

It looks as though the samples A and B are male and that D is female, with the rest being ambiguous.

  • A:1SWWBD
  • B:2SWWBD
  • D:R5#51

60°C

Aim - try to see if results are better at 60°C

method:

Follwing exact same protocol as here: [2] Master mix for all 8 samples plus -ve control(10 units of MM):

  • 25ul buffer (10x)
  • 20ul dNTPs
  • 15ul MgCl2
  • 1ul Taq
  • 25ul P2 primer (F)
  • 25ul P8 primer (R)
  • 129ul H2O

Tubes labelled as follows:

  • AX:1SWWBD
  • BX:2SWWBD
  • CX:R5#9
  • DX:R5#51
  • EX:R5#52
  • FX:R5#100 - (i htink i missed this one)
  • GX:Pink Gold
  • HX:banjo
  • -ve: 1ul water

PCR the same except annealing temp is 60°C (user 77 file 81).

results to be determined via agar gel E.Phoresis

55C

Same as above, samples AT,BT..etc

PCR anneal at 55°C


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