RNA denaturing gels

Glenn Dunshea via methods%40net.bio.net (by glenn.dunshea from gmail.com)
Mon Sep 24 19:14:10 EST 2007


SYBR gold is excellent for detecting both single stranded and double
stranded DNA and RNA. I do a 20 minute soak of gel running buffer, 50%
glycerol and 1-0.5x SYBR gold and get strong visualization of single
stranded DNA.

On 9/24/07, IanMc <zebedeeboy from hotmail.com> wrote:
>
> Hi Pow,
>
> We have used the RNA ladder from Ambion with no problem. But all our RNA
> work is done on an Agilent Bioanalyser. I don't have much experience with
> gels but I'm under the impression that EtBr binds less well to single
> strand
> vs double strand. So the protocol's use of a tonne of EtBr may have some
> basis. I would suggest two things, using a control plasmid (from the kit
> if
> Ambion supply one) and try to silver stain your gels rather than use EtBr.
> I
> don't know if there is a more RNA friendly fluorescent dye to use in place
> of EtBr, anyone?
>
> Ian Mc
>
> "Pow Joshi" <pow.joshi from gmail.com> wrote in message
> news:mailman.1022.1190403087.11350.methods from net.bio.net...
> > 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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