User:Mar/Notebook/2007-8-6
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Optimization of PCR cycles for genotyping (2)
Goal: shorten PCR cycling while preserving detection level
- prepared 8x20µL = 150µL of mix:
GreenGoTaq - 75µL primers - 7.5µL each water - 45µL rl117b (het) DNA - 7.5µL
- aliquoted : 4x5µL; 4x10µL; 4x20µL, added 10µL mineral oil to 5µL and 10µL tubes and frozen all tubes on dry ice
- started AWS10, AWS15 (4:07pm-5:33) and AWS30 (4:10pm-6:00) on sets of tubes, then frozen amplificates until next day
- started AWS with another set of tubes o/n, then frozen in the morning
- (next day) thawed and run gel
ramp time for PTC-200: up to 3°C/sec
actual time @ 20µL: 1°C/sec
programs used:
| AWS | AWS30 | AWS15 | AWS10 | |
|---|---|---|---|---|
| 94°C | 5' 3.82 1.58 1.20 1.08 | 5' | 5' | 5' |
| 94°C | 1' | 30" | 15" | 10" |
| 55°C | 2' | 30" | 15" | 10" |
| 72°C | 3' | 30" | 15" | 10" |
| cycles total | 30x | 30x | 30x | 30x |
| 72°C | 10' | 10' | 10' | 10' |
| total time [hr] @ 20µL | 3.82 | 1.58 | 1.20 | 1.08 |
