User:Stuart McKellar/Notebook/Bird Sex Testing/2012/12/01: Difference between revisions
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== | ==higher annealing temp== | ||
* | |||
Aim: test the p2/p8 and 2550/2178 primers at 65°C. | |||
Method: | |||
Sample 1: | |||
*A6: p2/p8 primers | |||
*B6: 2550/2178 primers | |||
Sample 2: | |||
*C6: p2/p8 primers | |||
*D6: 2550/2178 primers | |||
Negative control - no DNA (1ul h2O, 2550/2178 primers.) | |||
Master mix (5x): | |||
*12.5μL buffer 10x | |||
*12.5μL dNTPS | |||
*7.5μL MgCl<sub>2</sub> | |||
*62.5 H<sub>2</sub>O | |||
*1μL Taq | |||
*A6: p2/p8 primers (2.5μL each, 1μL DNA) + 18.5 Master Mix | |||
*B6: 2550/2178 primers (2.5μL each, 1μL DNA) + 18.5 Master Mix | |||
*C6: p2/p8 primers (2.5μL each, 1μL DNA) + 18.5 Master Mix | |||
*D6: 2550/2178 primers (2.5μL each, 1μL DNA) + 18.5 Master Mix | |||
total volume in each is 25μL. | |||
PCR: 94°C for 2m30s, 35 cycles of: 95°C 30s, 65°C for 30s, 72°C for 30s; final elongation 72°C for 5 mins. | |||
results to be determined via agar gel electrophoresis on 1% gel stained using gelgreen. | |||
Revision as of 20:34, 30 November 2012
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higher annealing tempAim: test the p2/p8 and 2550/2178 primers at 65°C. Method: Sample 1:
Sample 2:
Negative control - no DNA (1ul h2O, 2550/2178 primers.) Master mix (5x):
total volume in each is 25μL. PCR: 94°C for 2m30s, 35 cycles of: 95°C 30s, 65°C for 30s, 72°C for 30s; final elongation 72°C for 5 mins. results to be determined via agar gel electrophoresis on 1% gel stained using gelgreen.
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