RNA denaturing gels
(by pow.joshi from gmail.com)
Tue Sep 25 17:58:54 EST 2007
Thank you everyone for the input. I shall try the new methods for my RNA.
On 9/25/07, corona <customer from newsnet.com> wrote:
> >> > 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
> >> > 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
> >> > then
> >> > the bands I got were diffuse, and I could'nt really say what size.
> >> > 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
> >> > anything on the Ambion message kit, I'd appreciate it much.
> >> >
> >> > thank you,
> >> > pow
> formaldeyde gels are known to give fuzzy bands, but is often used because
> you can do northern blots with the gel afterwards. If your simply wanting
> size your RNA then I suggest you run a gloxal gel, which will give you a
> cleaner band. In both cases, you need to make sure all your solutions are
> RNase free and your gel tank and casting comb has been washed with a
> solution to kill RNases (e.g., alcoholic KOH) and then with RNase free
> to restore PH. Goodluck.
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