I strongly recommend that you first do RNase digestion in TE at 37C for at least 30 min. Then add 0.5% SDS and proteinase K (remains active for a long time) at 65C for up to overnight.

hi hannah... i'm not getting why r u using RNAse and proteinase together, first with TE and 0.5 % SDS add proteinase and incubate at 37 degree for 2 hrs, then add RNAse and then incubate at 55 degree for 2 hrs and finally go for phenol, chloroform step.

Thanks Prajna. I don't get it either which is why I'm asking!

I strongly recommend that you first do RNase digestion in TE at 37C for at least 30 min. Then add 0.5% SDS and proteinase K (remains active for a long time) at 65C for up to overnight.

Hannah, what we do is to digest first with proteinase K at 55C until the sample is clear, than, you inactivate the ptnase K at 95C for 10 min, because it will, in fact, inhibit your RNase. After you do that, you treat with RNase at 37C (I do for 1:30h) then, follow your protocol...

Thanks. You've confirmed what I thought was logical. I think I'll go with a two step process for the RNAse and Preoteinase and then do the chloroform ethanol steps.

Another reason for the two step: TE contains EDTA which sequesters Ca2+ and Ca2+ is needed for RNAse whereas proteinase K can function independently from Ca2+ to a degree...

I have found in literature that proteinase k does not inhibit rnase to the point where you should not add the two simultaneously, however, it is best to have the solvent as PBS for the reason I mentioned previously. If what you are doing seems to work I would not change your protocol at this point. Good luck!

I was wondering the same thing looking through the Invitrogen PureLink Genomic DNA Prep Kit. A quick search of PubMed yielded the article linked below, suggesting that RNase A and Proteinase K can peacefully coexist in a cellular digestion reaction.

http://www.ncbi.nlm.nih.gov/pubmed/153663