User:Stuart McKellar/Notebook/Bird Sex Testing/2012/11/23

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PCR optimisation

Yesterday failed to yield a single successful result. The two steps that were universal across all samples were number of cycles, an extended 72C period of 20 mins after 25 cycles, and 0.5U taq.

Aim: Today I will try with 3 different levels of taq and dna and run everything else the same as for the initial successful run. The tubes contents will be as follows:

  • 1 x Master Mix(uL):
  • 2.5 Buffer (10x)
  • 2.5 Forward (P2)
  • 2.5 Reverse (P8)
  • 2.5 dNTPs
  • 1.5 MgCl2
  • 10 H20

DNA From R5#9 (DNA 186.53 ng/ul)

Then as follows for each tube:

  • AA: 0.5ul DNA, 0.5U Taq; H20= 2.5
  • AB: 0.5ul DNA, 1.0U Taq; H20= 2.0
  • AC: 0.5ul DNA, 1.5U Taq; H20= 1.5
  • AD: 1.0ul DNA, 0.5U Taq; H20= 2.0
  • AE: 1.0ul DNA, 1.0U Taq; H20= 1.5
  • AF: 1.0ul DNA, 1.5U Taq; H20= 1.0
  • AG: 2.0ul DNA, 0.5U Taq; H20= 1.0
  • AH: 2.0ul DNA, 1.0U Taq; H20= 0.5
  • AI: 2.0ul DNA, 1.5U Taq; H20= 0.0
  • A-ve: 0.0ul DNA 1.0U Taq; H20= 2.5

All of the tubes had 24.5 ul total. They had 21ul of MM, instead of 21.5.


Visualised on a 1% gel. 500V, 75mA for about 30 minutes. Bars everywhere! Sample looks female.



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