RNA on denaturing gels
utsuxs from hotmail.com
(by utsuxs from hotmail.com)
Tue Jul 8 20:02:11 EST 2008
On Jun 27, 5:35 am, "Simone Marker" <mar... from rhrk.uni-kl.de> wrote:
> since I want to do a northern blot, I have to perform a denaturing agaose
> gel electrophorese with total RNA of Paramecium.
> My problem is, that I don't even get the typical bands (28S and 18S rRNA)
> for total eukaryotic RNA. I isolated the RNA with Trizol and prepared a 1,1%
> agarose gel with Formaldehyd (3-4ml 37% per 40ml gel solution) in a northern
> running buffer (20mM MOPS, 5 mM NaAc, 1mM EDTA). The run was at 50-70V.
> I denatured the RNA samples at 65°C for 5-10 min.
> I used a ssRNA ladder in its original purchased loading buffer (7M urea,
> ficoll,...), which showed a perfect run in this gel. In contrast, my RNA
> samples in this loading buffer showed a pattern of different bands with one
> very dominant (at ca 3500 nt).
> When I used another common loading buffer receipe(50% formamide, 25%
> formaldehyde,...), I got a smear and some weak bands. I can nearly exclude
> that the RNA that I used was not severely degraded, since I had it on a
> bioanalyser a few month ago (stored at -70°C).
> What might be wrong in my system?
> Thank you very much!
These are just random thoughts.
You sure you have RNA and not chewed up DNA?
Just to confirm, you got two bands/peaks on the bioanalyser?
I forgot where tRNA ends up but that could be the 3500nt band.
And contamination. Your ingredients and equipment, including your gel
box might contaminated with RNase which is hell to get rid of.
Wouldn't matter if your RNA is RNA and intact, you drop it in an RNase
contaminated environment, well so long RNA.
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