Smearing of the PCR Products - (Jun/07/2006 )
check your amplicon - length AT-GC secondury structures (hairpins)
GC-rich - betaine AT- rich - magnesium
ELONGATION time -- 25 seconds for 1 kb for Taq ( do not ovrhold at any!!!!!!! temperatures)
Try ammonium PCR buffer or use GoTaq Green flexy(promega) - it is robost and
resist wide range of magnesium concentrations
Calculate INPUT COPY NUMBER
if DNA is DAMAGED - Taq +Pfu ( Long PCR blend ) helps
Touchdown - Stepdown - Hotstart will help
Use cycler with ramping rates 3 C / sec or more under "tube control" heated lid
reaction volume - 20 mkl- PCR plastics from Abgene
-artem-