User:Stuart McKellar/Notebook/Bird Sex Testing/2012/11/27
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
Comparing primersAim: 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):
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)
-ve control
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:
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.
|