RNA denaturing gels
(by zebedeeboy from hotmail.com)
Mon Sep 24 04:54:08 EST 2007
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?
"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
> ergo, here are the details:
> I make a mRNA by the mMessage Machine kit from Ambion. The mRNA is
> 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
> of EtBr in the sample buffer, which ofcourse runs in the opposite
> and gave me a large background. I allowed it to run out of the gel....
> 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,
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