User:Mar/Notebook/2007-8-10

= 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
GreenGoTaq - 80µL primers - 8µL each water - 48µL rl117a (het) DNA - 8µL
 * prepared 8x20µL = 160µL of mix:
 * 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:

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