I'm extracting genomic bacterial DNA in reasonably large quantities and I need it to be high quality too.
The final step in the protocol I have been given incubates overnight in TE with 0.5% SDS, and 0.5mg/ml each of RNAse and Proteinase. Will the proteinase damage the RNAse?
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.
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
As usual, classic books also got the answer. You can find DNA extraction protocols in the Molecular Cloning books and see that they also mix RNAse and Proteinase K. RNAse does not work at high concentrations of SDS, though, so you have to increase its concentration if you want to use it in a solution containing 0.5% of SDS (20 ugr/mL final concentration)
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