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Proteinase K - How to use it? (Jan/24/2007 )

I have a DNA suspension, which I suspect contains proteinase and therefore no PCR products were obtained due to the degradation of my Taq polymerase by this proteinase. What I would like to try now is to degrade this proteinase with proteinase K prior to PCR.

I'm not sure how to use proteinase k, in terms of how much to use with a particular amount of DNA and the protocol such as whether incubation, centrifugation or ethanol/isopropanol precipitation is required. Please advice.


I have the pretty much similar encounter.

I usually use Proteinase K together with alcohol precipitation. So basically, this can get rid of the protein and also purify DNA prior to PCR. I used use about 0.5 - 1ul of Proteinase K (depends on your DNA). If you can see some protein (white suspension), use more proteinase K. My proteinase K is from NEB. I can't remember the concentration. Sorry about that.

Then incubate it for 15 minutes at 55 Celcius. After that, you can do your ethanol precipitation. Usually work for me. Just my 2 cents worth opinion. Hope it helps. wink.gif


So, to remove a protease you are adding a protease? That's fine, except you have to remove the protease. The usual approach to this problem would be phenol/chloroform extraction of your DNA sample, followed by chloroform extraction and ethanol precipitation. If you go the protease route, it might be easier to treat your sample with PMSF to inactivate any (most) proteases present. If you digest with proteinase-K, then I would suggest PMSF treatment prior to adding any enzymes I expected to be active.