User:Mar/Notebook/2007-8-28
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Optimization of PCR cycles for genotyping (6, part 5)
Goal
- shorten PCR cycling while preserving detection level
- testing 2-step PCR
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.
Acc. to Eppendorf BioNews Application Notes (Lopez & Prezioso, A better way to optimize: Two-step gradient PCR, Sept 2001):
- test cycle: 94°C/2' - (94°C/15" - 50-70.5°C/30")x30 - 72°C/1'
- screen for intensities of specific vs. nonspecific amplicons
Protocol
- cycle: 94°C/5' - (94°C/30-60' - 55°C/3045-60")x30 - 72°C/10' {60", 30"} x {60", 45"}
- prepared 4x10µL =< 50µL of mix:
GreenGoTaq - 25µL 3 primers - 2.5µL each water - 15µL rl117a (het) DNA - 52.5µL
- aliquoted : a 10µL, added 10µL mineral oil and frozen all tubes on dry ice
- cycling started, then amplificates frozen
- thawed and run gel on 10µL 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
- most balanced doublet 60"/30" and 30"/60"
- the strongest doublets (almost the same): 30"/60" and 60"/60"
- ergo:
- shortening of annealing time from 60" down to 30" greatly decreases yield
- shortening of denaturiation time down to 30" does not affect yield, although may slightly affect balance
Future directions
- recheck {60", 30"} x {60", 45"}