User:Stuart McKellar/Notebook/Bird Sex Testing/2012/12/10

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PCR with positive controls

Aim: See if we can identify the positive controls using both primers.

Materials and methods: We will be running 5 x PCR reactions: male and female with p2/p8, male and female with 2550/2178 primers, and a negative control. We will run it at 60°C.

reaction mix:

  • 2.5ul F primer
  • 2.5ul R primer
  • 2.0ul dNTPs
  • 0.5u Taq
  • 1ul DNA
  • 2.5ul 10x buffer
  • 1.5ul MgCl2
  • 13ul H2O

Master Mix: 12.5 F 12.5 R 0.5 Taq 10 dNTPs 12.5 Buffer 7.5 MgCl2 65ul H2O

Negative control used p2/p8 primers as they are the most likely ones to work at these temps. Primers were not added to MM, but instead to each individual tube.

repeated experiment at 58°C.

ran PCR for 35 cycles, annealing temperature is 60°C

looks like faint bands are appearing. I may need to alter the times a little bit, maybe extend the amount of time for elongation. Two bands were distinguishable on the 58 and 60C PCRs when viewed on a 1% gel.

The second one which was 58C annealing temp ran on a 1% gel. I want to try 2550 primer set at 55C today. I might run the 2550 samples on the tawny's and the eclectus.