User:Mar/Notebook/2007-8-17

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Contents

Optimization of PCR cycles for genotyping (6)

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/1' - 50-70.5°C/1')x30 - 72°C/10' (annealing every 2.5°C)
  • prepared 11x10µL =< 120µL of mix:
GreenGoTaq - 60µL
3 primers - 6µL each
water - 36µL
rl117a (het) DNA - 6µL
  • aliquoted : a 10µL, added 10µL mineral oil and frozen all tubes on dry ice
  • started EP50 and EP55, then frozen
  • started EP60 and EP65, then frozen
  • started EP70 and EP75, then frozen
  • started EP52.5 and EP57.5, then frozen
  • started EP62.5 and EP67.5, then frozen
  • started EP72.5 then frozen
  • 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

  • no bands, except for a hint in 50°C annealing

Future directions

  • run the same program for annealing temp. range 40°C-55°C
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