User:Stuart McKellar/Notebook/Bird Sex Testing/2012/11/27

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Comparing primers

Aim: I am going to see if the 2550 primer set will amplify under the same conditions as the P2/P8 primers, and if so check if the results are more reliable.

The PCR product has a greater size difference I believe.

Methods:

Master mix for all 8 samples plus -ve control(10 units of MM):

  • 25ul buffer (10x)
  • 20ul dNTPs
  • 15ul MgCl2
  • 2ul Taq
  • 130ul H20

Then prepare tubes labelled as follows: P2 Primers(all contain 1ul of DNA of sample and 2.5ul of each relevant primer)

  • PA Sample 1 - 1swwbd
  • PB Sample 2 - r5 # 9
  • PC Sample 3 -r5 # 100
  • PD Sample 4 - pink gold

2550 Primers (all contain 1ul of DNA of sample and 2.5ul of each relevant primer)

  • 25A Sample 1 - 1swwbd
  • 25B Sample 2 - r5 # 9
  • 25C Sample 3 -r5 # 100
  • 25D Sample 4 - pink gold

-ve control

  • Add 2.5ul of p2 primer, 2.5ul p8 primer and 1ul of water.

Then add 19ul of master mix to each tube.

Run on the PCR - 95C for 2.5mins; 25 cycles of 30s each: 94,52,72; then final elongation of 72C for 5mins.

Run on 1% Gelgreen Gel.

Results:

  • Lane 1: - 1swwbd P2 - PA - One thick band
  • Lane 2: - 1swwbd 2550F - 25A - Non specific bands
  • Lane 3: - r5 # 9 P2 - PB - Non specific bands
  • Lane 4: - r5 # 9 2550F - 25A - Non specific bands
  • Lane 5: Ladder 3Kb - Ladder-like
  • Lane 6: - r5 # 100 P2 - PC - One thick band
  • Lane 7: - r5 # 100 2550F - 25A - Non specific bands
  • Lane 8: - Negative Control - one small band (presumably primers)

Discussion:

Out of all the samples, the P2/P8 primers gave 2 usable results out of 3 samples tested under these conditions. It is obvious that the annealing temperature needs to be raised on the 2550F/2178R primer combo. The P2/P8 primers, whilst yielding a result, will only have very small differentiation between alternate products. I should up the annealing temperature and try with the 2550F/R primers tomorrow.



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