Adding 1/100 Rnase A to 500ul CTAB buffer when extracting gDNA from plant powder often result in decrease of DNA concentration in the end. I wonder if the Rnase A is not pure enough containing Dnase contaminate or is something wrong elsewhere ?
Dear Luke,
It would be quite informative to have your RNAse preparation protocol and your CTAB extraction protocol, if you are interested in finding how what's happened.
RNAse is usually prepared by boiling at 100C for 10 min because is quite thermoestable, whilst the DNAse gets quickly denatured (see Sambrook Maniatis book, e.g.)
Also, how have you quantified your DNA concentration with/without RNAse treatment? I know it's obvious, but you cannot really distinguish DNA from RNA with spectrophotometry. Therefore, when you RNAse-treat samples you degrade the RNA into nucleotides or small oligos, and depending on how you precipitate afterwards you might not recover that degraded RNA, resulting in a lower nucleic acid concentration in comparison with the non-RNAse treated sample.
Cheers,
Ruben
Really? It's intereting. But I bet it's more likely the DNase contamination.
i think your enzyme is contaminated
any how when u give long term RNase A treatment their may be loss of DNA in small amount.
Yes you are right, there is obvious contamination of Dnase. You can solve this problem by heating the Rnase to 60 0C. Heating will degrade Dnase. Rnase is thermostable. Some researcher also suggests the addition of small quantity of EDTA.
I agree that it is probably DNase contamination. I would never use RNase without routinely heat treating it.at 90 degrees for 5 minutes. You can do this on a concentrated stock and then store it in the freezer.
Just an additional thought. I don't know the exact steps in your procedure but it could be that you have not removed all endogenous nuclease from your prep before you do the incubation and it is the act of doing an incubation that is the problem. I would try to make the DNase tratment as late as possible in the procedure.
Dear Luke,
It would be quite informative to have your RNAse preparation protocol and your CTAB extraction protocol, if you are interested in finding how what's happened.
RNAse is usually prepared by boiling at 100C for 10 min because is quite thermoestable, whilst the DNAse gets quickly denatured (see Sambrook Maniatis book, e.g.)
Also, how have you quantified your DNA concentration with/without RNAse treatment? I know it's obvious, but you cannot really distinguish DNA from RNA with spectrophotometry. Therefore, when you RNAse-treat samples you degrade the RNA into nucleotides or small oligos, and depending on how you precipitate afterwards you might not recover that degraded RNA, resulting in a lower nucleic acid concentration in comparison with the non-RNAse treated sample.
Cheers,
Ruben
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