DNA template for a mouse cell suspension , in 20 mM Tris, pH 7.9, 0.5% Tween 20, 2 mM EDTA,
65 degrees, 1 hour. At 65 degrees, proteinase K also eats up itself, so the DNA is PCR-ready.
DO DON'T
LA-PCR Tip #10 Insist on speed and 30 sec.
Use nice long extension steps. or 4 minute extensions.
10 minutes for 2 to 5 kb,
up to 22 minutes for 20-35 kb.
For shorter targets (up to 2 kb) we find that we can get by with 8-10 times less DNA polymerase
(KlentaqLA; 2-3 times less for Taq and TaqLA) if we use extension steps that are 2 to 5 times longer. That
is, 10-20 minutes instead of 2-4 minutes.
DO DON'T
LA-PCR Tip #11 Extend at 72 C.
Extend at 68 C, or This may be the optimum
even lower, such as 62-65. for a DNA polymerase assay
using nicked calf-thymus DNA
as template, but that is not PCR.
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