How does RNA affect ethidium bromide fluorescence?

Question

When trying to quantify DNA in a plasmid prep, and using EtBr as a fluorescent dye, will RNA in the sample greatly alter the values obtained?

Answer 1

Yes.  RNA fluoresces more strongly than DNA and will blow away any readings you get from DNA. You can treat your sample with some RNase before you read your sample on a spec.  If you are running your sample out on a gel to visualize it, you should add a small amount of RNase to the sample and incubate it for a few minutes before loading and running it.

Answer 2

If you do your plasmid prep properly, even if you don't  use RNase, you should have no problem with large amounts of contaminating RNA.

If you are quantifying visually on a gel, though, make sure that you are looking at bands of DNA and not RNA.  You might see two fairly bright bands of RNA if you didn't do your prep correctly.  In any case, as long as you include a standard of known DNA concentration, like some lambda phage DNA, you should have no problem.