User:Stuart McKellar/Notebook/Bird Sex Testing/2012/11/27: Difference between revisions
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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. | |||
Revision as of 00:43, 27 November 2012
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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.
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