User:Stuart McKellar/Notebook/Bird Sex Testing/2012/12/02

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Trying all loris with 65°C PCR - p2/p8

Aim: To see if at 65°C i can differentiate males from females.

Method: 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 H2OH20

Tubes labelled as follows:

  • A:1SWWBD
  • B:2SWWBD
  • C:R5#9
  • D:R5#51
  • E:R5#52
  • F:R5#100
  • G:Pink Gold
  • H:banjo
  • -ve: 1ul water

Add 1ul of relevant DNA to each tube and 24ul of MM.

PCR: 94°C for 2m30s, 35 cycles of: 95°C 30s, 65°C for 30s, 72°C for 30s; final elongation 72°C for 5 mins( actually went 10 mins by accident).

results to be determined via agar gel electrophoresis on 1% gel stained using gelgreen.



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