User:Mar/Notebook/2007-8-6

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Optimization of PCR cycles for genotyping (2)

Goal: shorten PCR cycling while preserving detection level

  • prepared 8x20µL = 150µL of mix:
GreenGoTaq - 75µL
primers - 7.5µL each
water - 45µL
rl117b (het) DNA - 7.5µL
  • aliquoted : 4x5µL; 4x10µL; 4x20µL, added 10µL mineral oil to 5µL and 10µL tubes and frozen all tubes on dry ice
  • started AWS10, AWS15 (4:07pm-5:33) and AWS30 (4:10pm-6:00) on sets of tubes, then frozen amplificates until next day
  • started AWS with another set of tubes o/n, then frozen in the morning
  • (next day) thawed and run gel


ramp time for PTC-200: up to 3°C/sec

actual time @ 20µL: 1°C/sec

programs used:

AWS AWS30 AWS15 AWS10
94°C 5' 3.82 1.58 1.20 1.08 5' 5' 5'
94°C 1' 30" 15" 10"
55°C 2' 30" 15" 10"
72°C 3' 30" 15" 10"
cycles total 30x 30x 30x 30x
72°C 10' 10' 10' 10'
total time [hr] @ 20µL 3.82 1.58 1.20 1.08
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