User:Mar/Notebook/2007-8-27

= Optimization of PCR cycles for genotyping (6, part 4) =

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
GreenGoTaq - 25µL 3 primers - 2.5µL each water - 15µL rl117a (het) DNA - 52.5µL
 * cycle: 94°C/5' - (94°C/30-60' - 55°C/30-60")x30 - 72°C/10' (denaturation/annealing x 30/60")
 * prepared 4x10µL =< 50µL of mix:
 * aliquoted : a 10µL, added 10µL mineral oil and frozen all tubes on dry ice
 * cycling started, then amplificates 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:

Results

 * most balanced doublet 60"/30" and 30"/60"
 * the strongest doublets (almost the same): 30"/60" and 60"/60"
 * ergo:
 * shortening of annealing time from 60" down to 30" greatly decreases yield
 * shortening of denaturiation time down to 30" does not affect yield, although may affect balance

Future directions

 * recheck {60", 30"} x {60", 45"}