User:Mar/Notebook/2007-8-10

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Optimization of PCR cycles for genotyping (3)

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 8x20µL = 160µL of mix:
GreenGoTaq - 80µL
primers - 8µL each
water - 48µL
rl117a (het) DNA - 8µL
  • aliquoted : 3x5µL; 3x10µL; 5x20µL, added 10µL mineral oil to 5µL and 10µL tubes and frozen all tubes on dry ice
  • started AWS01, AWS10 (4:07pm-5:33) and AWS20 (4:10pm-6:00) on sets of tubes, then frozen
  • started AWS (with 2 tubes of 20 µL (deviation: it was supposed to be AWS + AWS60, one of each), frozen
  • (next Monday) 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

AWS 20µL - predictably strong balanced doublet
AWS20 - all 3 volumes equal in intensity, overall slightly weaker than AWS, lower (shorter) band slightly weaker
AWS10 + AWS01 - all 3 volumes in both cycles ~equally intense, lower (shorter) band much weaker

Future directions

  • drop AWS > 20min for a while
  • drop 20µL - they don't do any better than 10µ
  • run comparison of 52.5 - 55.0 - 57.5°C annealing
  • run comparison of 20µL vs. 10µL ramping
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