RNA denaturing gels

Sudheendra Rao N R via methods%40net.bio.net (by sudhee26 from gmail.com)
Wed Oct 17 08:02:19 EST 2007


Pow,
I hope u have run the "new" RNA gel by now. I want to share few thing with
you.
I use RNA for RTPCR so i just need to check for the integrity. I dont use
denaturing gels and so i use regular DNA agarose gel (TAE buffer,
2microLEtBr) and i get 3 expected bands including 5s one.
So depending on why u need a RNA gel, u can go for either denaturing and all
those excercises or stick to regular DNA gel..
Hope it helps.

Sudheendra.


On 9/26/07, Pow Joshi <pow.joshi from gmail.com> wrote:
>
> Thank you everyone for the input. I shall try the new methods for my RNA.
>
> Pow
>
> 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
> > 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
> >
> > 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
> > to
> > 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
> > water
> > to restore PH. Goodluck.
> >
> >
> >
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> >
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