I have never seen a PCR that worked better at 72 than at 68, but I always test long targets. For
long PCR, 68 is definitely better. For any PCR, the recommendation of 72 by some companies is based
on the optimum for Taq DNA polymerase activity in a standard assay that is not PCR. This assay, which
dates from the pioneering first paper about DNA polymerase (Lehman et al., 1958) is a based on
incorporation at nicks, gaps, and/or staggered ends of calf thymus DNA that has been partially degraded
and heated to form poorly defined structures. To optimize PCR, I suggest that PCR reactions are more
appropriate.
DO DON'T
LA-PCR Tip #12 Dilute the enzyme mixture
Add enzyme from full-strength in anything but reaction mix
stock, to a mix for multiple reactions. with KLA buffer in there first,
except for very short periods.
This is just generally good advice for enzyme stability. Enzymes generally more stable in higher
salt and at higher concentration. If you must dilute the enzyme, for KlentaqLA the storage buffer is 50%
glycerol (v/v; 63% w/v), 222 mM ammonium sulfate, 20 mM Tris-HCl pH 8.55, mM EDTA, 10 mM
mercaptoethanol, 0.5% IGEPAL, 0.5% Tween 20.
DO DON'T
LA-PCR Tip #13 Heat the RNA to 90 or so.
Assemble your RT reaction Unless there is excess EDTA,
on ice, and warm to 45 after Mg and other metals will cause
adding reverse transcriptase last. RNA breakdown.
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