User:Mar/Notebook/2007-8-23

= Optimization of PCR cycles for genotyping (6, part 3) =

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 - 35µL 3 primers - 3.5µL each water - 21µL rl117a (het) DNA - 3.5µL
 * cycle: 94°C/5' - (94°C/1' - 45-65°C/1')x30 - 72°C/10' (annealing every 5°C)
 * prepared 6x10µL =< 70µL of mix:
 * aliquoted : a 10µL, added 10µL mineral oil and frozen all tubes on dry ice
 * started EP40 and EP45, then frozen
 * started EP50 and EP55, then frozen
 * started EP42.5 and EP7.5, then frozen
 * started EP52.5 and EP57.5, then 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

 * balanced doublet @ 55°C confirmed, though a little weaker then best unbalanced doublet (55°C)
 * annealing at the temp. < 55°C yields gradually weaker doublets, with longer band always weaker than shorter
 * annealing at the temp. 60°C yields only longer band
 * annealing at the temp. 65°C yields no bands

Future directions

 * recheck the same program for annealing temp. range 53 - 54 - 55 - 56 - 57 - 58°C