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RNA on denaturing gels

RNA on denaturing gels - Protocols and Methods Forum

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  #1  
Old 06-27-2008, 09:35 AM
Simone Marker
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Default RNA on denaturing gels



Hi,

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 65C 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 -70C).

What might be wrong in my system?
Thank you very much!
simone


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  #2  
Old 07-09-2008, 01:02 AM
utsuxs@hotmail.com
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Default RNA on denaturing gels

On Jun 27, 5:35 am, "Simone Marker" <[Only registered users see links. ]-kl.de> wrote:

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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  #3  
Old 07-09-2008, 10:54 AM
Peter Ellis
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Default RNA on denaturing gels

On Tue, 8 Jul 2008, [Only registered users see links. ] wrote:

Transfer RNAs are ~75 bp, so.... no.


Yes, but the OP says his ssRNA ladder ran fine, which argues against
degradation in the gel.

To the OP - are you sure what size ribosomal bands you're expecting from
Paramecium? I strongly doubt it'll be the exact same pattern/size you'd
expect from mammalian cells.

Peter
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