Methods Digest, Vol 28, Issue 21

Barbara MacGregor via methods%40net.bio.net (by bmacgreg from unc.edu)
Sat Sep 22 15:22:05 EST 2007


Hi Pow,

I would recommend either a denaturing acrylamide gel - about a 10%  
minigel should do it - or else a glyoxal/agarose gel, if you're not  
set up for acrylamide. The acrylamide gives you sharper bands and  
better sensitivity, in my experience. I could email you detailed  
protocols for either or both if you like.

Barbara

On Sep 22, 2007, at 1:00 PM, methods-request from oat.bio.indiana.edu wrote:

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>    1. RNA denaturing gels (Pow Joshi)
>
> From: "Pow Joshi" <pow.joshi from gmail.com>
> Date: September 21, 2007 2:29:16 PM EDT
> To: Methods from magpie.bio.indiana.edu
> Subject: RNA denaturing gels
>
>
> Hello Experts,
>
> I am trying to run a RNA gel, (which I had run a very long time  
> ago, and
> time tends to erode all  information from my chamaeleonic nervous  
> system!),
> ergo, here are the details:
> I make a mRNA by the mMessage Machine kit from Ambion.  The mRNA is  
> capped,
> with a poly A tail of about 19 bases. The insert size is ~2kb, and  
> so I
> expect a band around 1.9-2.1 kb.
> When I regular agarose gel, I see some band ~1kb. I have two other  
> inserts
> about the same size, and I do'nt really see much with them.
> I tried running a folmaldehyde gel, and the protocol I followed had  
> a tonne
> of EtBr in the sample buffer, which ofcourse runs in the opposite  
> direction,
> and gave me a large background. I allowed it to run out of the  
> gel.... then
> the bands I got were diffuse, and I could'nt really say what size. The
> marker was another story: this one from NEB, which did'nt show up  
> at all.
>  I obviously don't know too much about RNA gels, and if anyone  
> could shed
> some light on how to better the technique or interpret results or even
> anything on the Ambion message kit, I'd appreciate it much.
>
> thank you,
> pow
>
>
>
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