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Does Proteinase K digest RNAse in lysis buffer?


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#1 biologg

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Posted 09 November 2012 - 02:27 AM

Hello everyone,

i have always been wondering something, but never quite got a definite answer. Many lysis buffer recipes for DNA extraction contain both Prot K and RNAse. However, it makes no sense to me to add both at the same time, as Prot K degrades RNAse as well, does it not? So what is the point to add RNAse to get rid of your RNA, when Prot K is also present in the reaction? Does it make any difference if you add the RNAse just before you add the lysis buffer to your cells/sample? Maybe in this way the RNAse will degrade the RNA molecules (or at least a large amount of them) before the Prot K had the chance to degrade the RNAse? Some suggest adding RNAse first, but if you add it before the Prot K, then you risk DNA digestion by DNAses. Can someone clarify this for me? Thank you!

#2 Curtis

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Posted 09 November 2012 - 04:24 AM

Yes, prot K degrades RNAses. It's better to add RNAse first and then prot K.

Read this: http://www.ncbi.nlm....v/pubmed/153663

You are not the first person to ask this question.
http://www.protocol-...osts/21549.html

But also keep in mind that digestion of DNA by DNAse (in contaminated samples, tips, microtube) is not as rapid as digestion of RNA with RNAses. Therefore, you don't have to worry about that and you have time to add your prot k and RNAse. Also, prot k might need time to degrade RNAse, so even if you add them together, RNAse A will digest your RNA before prot k digests it. You need to realize you are adding non-cellular RNAse A and prot k. They are excess in your sample.

You can also try this by yourself. Extract total RNA and prepare these samples:

1- total RNA
2 - total RNA + RNAse A, incubate (10-30 min) + prot K
3 - total RNA + prot k + RNAse

run on gel and check if you see any RNA smear or 18s/28s bands in the 3rd sample. If you do then prot k degrades your RNAse, although I doubt it will completely do that.

#3 biologg

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Posted 09 November 2012 - 05:41 AM

Thank you, Curtis!




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