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.