User:Stuart McKellar/Notebook/Bird Sex Testing/2012/12/01
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(Autocreate 2012/12/01 Entry for User:Stuart_McKellar/Notebook/Bird_Sex_Testing) |
Current revision (01:31, 1 December 2012) (view source) (→higher annealing temp) |
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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( actually went 10 mins by accident). | ||
| + | |||
| + | results to be determined via agar gel electrophoresis on 1% gel stained using gelgreen. | ||
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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( actually went 10 mins by accident). results to be determined via agar gel electrophoresis on 1% gel stained using gelgreen.
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