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:
    1. shortening of annealing time from 60" down to 30" greatly decreases yield
    2. shortening of denaturiation time down to 30" does not affect yield, although may slightly affect balance

Future directions

  • recheck {60", 30"} x {60", 45"}