User:Mar/Notebook/2007-8-23

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Contents

Optimization of PCR cycles for genotyping (6, part 3)

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' - 45-65°C/1')x30 - 72°C/10' (annealing every 5°C)
  • prepared 6x10µL =< 70µL of mix:
GreenGoTaq - 35µL
3 primers - 3.5µL each
water - 21µL
rl117a (het) DNA - 3.5µL
  • aliquoted : a 10µL, added 10µL mineral oil and frozen all tubes on dry ice
  • started EP40 and EP45, then frozen
  • started EP50 and EP55, then frozen
  • started EP42.5 and EP7.5, then frozen
  • started EP52.5 and EP57.5, then 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

  • balanced doublet @ 55°C confirmed, though a little weaker then best unbalanced doublet (55°C)
  • annealing at the temp. < 55°C yields gradually weaker doublets, with longer band always weaker than shorter
  • annealing at the temp. 60°C yields only longer band
  • annealing at the temp. 65°C yields no bands

Future directions

  • recheck the same program for annealing temp. range 53 - 54 - 55 - 56 - 57 - 58°C
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