User:Mar/Notebook/2007-8-13

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Optimization of PCR cycles for genotyping (4)

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 9x10µL =< 100µL of mix:
GreenGoTaq - 50µL
3 primers - 5µL each
water - 30µL
rl117a (het) DNA - 5µL
  • aliquoted : a 10µL, added 10µL mineral oil and frozen all tubes on dry ice
  • started AWS20 @ 55°C, and AWS20 @ 52.5°C, then frozen
  • started AWS10 @ 55°C, and AWS10 @ 52.5°C, then frozen
  • started AWS01 @ 55°C, and AWS01 @ 52.5°C, then frozen
  • started AWS20 @ 57.5°C, and AWS10 @ 57.5°C, then frozen (deviation: both were run on AWS10 @ 57.5°C)
  • started AWS01 @ 57.5°C o/n, then frozen
  • (next day) 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

  • yield decreases with cycle time increase
  • AWS20: max yield at 55°C, but at 52.5°C bands are more balanced
  • AWS10: max yield at 52.5°C, at higher annealing temp. shorter band disappears
  • 55°C: AWS20 >> AWS10
  • 52.5°C: AWS20 < AWS10

Future directions

  • run {52.5°C vs. 55°C} x {AWS20 vs. AWS10} in duplicates

Mentoring

Taught thick sectioning to John

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