September 13, 2007 at 1:12 am #8224
I have a problem to solve, and I have few ideas how to approach it, but maybe you Guys have better solutions?
I have two samples, one of them contains RNA, while the other DNA. I don’t know which one is which. What are the possible ways to differentiate between them?
1) absorbance measurement A260/A280 for DNA 1.8, for RNA 2.0, pretty lame
2) RNaze A treatment which would digest RNA leaving my DNA sample intact, simple agarose gel would show me which is which
3) DNase digestion leaves RNA intact cleaves DNA, similar approach to the previous one ❗
4) restriction endonuclease, cuts just dsDNA, leaving RNA intact…
Any other ideas?
September 13, 2007 at 2:04 am #75981
how long are they?
September 13, 2007 at 5:37 am #75983MrMisteryParticipant
i may be wrong, but from what i know, EtBr only interacts with DNA, not RNA. If this is indeed true then put EtBr and stick in into the electrophoresis machine.
Also, PCR would deffinetly work in amplifying only the DNA, not the RNA
September 13, 2007 at 6:39 am #75992
No, etbr works on RNA too. In PCR’s we’d see a solid signal and then a fuzzier one further down stream.
September 13, 2007 at 8:57 am #75995MrMisteryParticipant
ok, like i say i wasn’t sure. but pcr would work
September 13, 2007 at 9:43 am #75997SororSaudadeParticipant
If you can spare some of you sample, I think the RNase or DNase treatment could be a very good and simple way of knowing that.
I’m not an expert though..
September 13, 2007 at 1:26 pm #75999canalonParticipantquote SororSaudade:
However lame solution 1 is not bad either if you cannot afford to lose the samples. of course this suppose a good purity of the samples. So I second SororSaudade if you can afford to do it (and I suspect you can, since this sounds more like lab/homework question than a real life experiment).
September 13, 2007 at 2:18 pm #76000
I’m sure EtBr intercalate with secondary structures of RNA, so that wouldn’t work for my purpose…
How about the use of restriction enzymes? Do you guys know the enzyme which would cut almost every kind of DNA leavind RNA intact?
September 13, 2007 at 5:13 pm #76003blcr11Participant
DNase I will work and so will RNase A, but make sure you use quality enzymes; no DNase contamination in the RNase A and vice versa. RNA is more sensitive to alkaline hydrolysis compared to DNA (which is why it is alkaline lysis you use to purify plasmid DNA). RNA usually runs way out front of typical plasmid DNA or restriction fragments on agarose gels and shows up as a fuzzy spot at really low MW—you can even run it off the gel if you run too long—after staining with EtBr. If there’s a lot of RNA contamination—like if you forget to add RNase to the lysis buffer when preparing your plasmid DNA—the RNA staining will be very intense and raise the background staining in the entire well. I think there are some RNA-specific dyes around—Northerns were once stained with acridine orange(?) or something like that if I remember correctly. You can do the 260/280 ratio thing, but it is often hard to interpret. The rule of thumb that “ratios of 2 or more imply RNA” is often correct, but not always.
September 15, 2007 at 2:16 am #76018jesusfreak0318Participant
one of them (DNA I believe) has thymine while the other (i think it’s RNA) has something else
September 15, 2007 at 2:23 am #76019
September 17, 2007 at 10:54 pm #76093
that’s a good observation!
September 18, 2007 at 6:44 am #76105najamParticipant
this can simply done by ultra centrifugation.DNA as a denser lay down and RNA due to its light weight becomr up to the surface.
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