PCR and Proteinase K ?

Markus Schneemann schneema at cmgm.Stanford.EDU
Thu Aug 27 13:56:38 EST 1998


Hi all:

We use Proteinase K (Boeringer) together with triton X100 for digestion of
tissue samples before PCR for specific bacterial genes (16S and beyond..).
Digestion buffer is:
50mM KCL
10mM Tris, pH8.3
1% Triton X100
Proteinase K (20mg/mL stock) end-conc.: 200ng/ul
(total volume 50ul - 10ug Proteinase K total)
Conditions are: 55 C / 2 hours vs. O/N

1) Does Proteinase K contain sufficient residual amounts of DNASe to
substantially reduce the DNA-content of our samples ?

2) Does it make a difference to digest for 2 hours or overnight at 55 C ?

In other words, could it be that we loose our template DNA by prolonged
incubation or a impure DNAse-containing Proteinase K.

If yes - do you know a trustworthy, pure, DNAse-free Proteinase K for our
purposes ?

Cheers,

Markus





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