User:Stuart McKellar/Notebook/Bird Sex Testing/2012/11/27
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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.
Master mix for all 8 samples plus -ve control(10 units of MM):
Then prepare tubes labelled as follows: P2 Primers(all contain 1ul of DNA of sample and 2.5ul of each relevant primer)
2550 Primers (all contain 1ul of DNA of sample and 2.5ul of each relevant primer)
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.
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.