User:Mar/Notebook/2007-8-13
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Optimization of PCR cycles for genotyping (4)
Goal: shorten PCR cycling while preserving detection level
Technical considerations
Acc. to Eppendorf's Tech Support pages:
- denaturation: min 1" (for 20µL) or 5" (for 50µL)
- annealing: 10-20" usually adequate
- extension: ~50/sec which yields 8sec for reeler, but keep in mind that during annealing (at 55°C), Taq extends too, ith ~25/sec speed.
Protocol
- prepared 9x10µL =< 100µL of mix:
GreenGoTaq - 50µL 3 primers - 5µL each water - 30µL rl117a (het) DNA - 5µL
- aliquoted : a 10µL, added 10µL mineral oil and frozen all tubes on dry ice
- started AWS20 @ 55°C, and AWS20 @ 52.5°C, then frozen
- started AWS10 @ 55°C, and AWS10 @ 52.5°C, then frozen
- started AWS01 @ 55°C, and AWS01 @ 52.5°C, then frozen
- started AWS20 @ 57.5°C, and AWS10 @ 57.5°C, then frozen (deviation: both were run on AWS10 @ 57.5°C)
- started AWS01 @ 57.5°C o/n, then frozen
- (next day) thawed and run gel on 5µL loads
ramp time for PTC-200: up to 3°C/sec (actual time @ 20µL: 1°C/sec)
programs used:
| AWS | AWS60 | AWS20 | AWS10 | AWS01 | |
|---|---|---|---|---|---|
| 94°C | 5' | 5' | 5' | 5' | 5' |
| 94°C | 1' | 1' | 20" | 10" | 1" |
| 55°C | 2' | 1' | 20" | 10" | 1" |
| 72°C | 3' | 1' | 20" | 10" | 1" |
| cycles total | 30x | 30x | 30x | 30x | 30x |
| 72°C | 10' | 10' | 10' | 10' | 10' |
| total time [hr] @ 20µL | 3.82 | 2.32 | 1.32 | 1.08 | 1.00 |
Results
- yield decreases with cycle time increase
- AWS20: max yield at 55°C, but at 52.5°C bands are more balanced
- AWS10: max yield at 52.5°C, at higher annealing temp. shorter band disappears
- 55°C: AWS20 >> AWS10
- 52.5°C: AWS20 < AWS10
Future directions
- run {52.5°C vs. 55°C} x {AWS20 vs. AWS10} in duplicates
Mentoring
Taught thick sectioning to John
