User:Mar/Notebook/2007-8-10
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Optimization of PCR cycles for genotyping (3)
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 8x20µL = 160µL of mix:
GreenGoTaq - 80µL primers - 8µL each water - 48µL rl117a (het) DNA - 8µL
- aliquoted : 3x5µL; 3x10µL; 5x20µL, added 10µL mineral oil to 5µL and 10µL tubes and frozen all tubes on dry ice
- started AWS01, AWS10 (4:07pm-5:33) and AWS20 (4:10pm-6:00) on sets of tubes, then frozen
- started AWS (with 2 tubes of 20 µL (deviation: it was supposed to be AWS + AWS60, one of each), frozen
- (next Monday) 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
AWS 20µL - predictably strong balanced doublet AWS20 - all 3 volumes equal in intensity, overall slightly weaker than AWS, lower (shorter) band slightly weaker AWS10 + AWS01 - all 3 volumes in both cycles ~equally intense, lower (shorter) band much weaker
Future directions
- drop AWS > 20min for a while
- drop 20µL - they don't do any better than 10µ
- run comparison of 52.5 - 55.0 - 57.5°C annealing
- run comparison of 20µL vs. 10µL ramping