RNA denaturing gels

Pow Joshi via methods%40net.bio.net (by pow.joshi from gmail.com)
Wed Oct 17 11:54:17 EST 2007


On 10/17/07, Sudheendra Rao N R <sudhee26 from gmail.com> wrote:
>
> 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.
>


Thanks Sudheendra...... I have usd regular gels, however, I wish to
ascertain the integrity of mRNA, which I make using the Ambion kit. The
regular agarose gels seem to have some issues with the size assessment since
the RNa would have some scondary structure. I have now resorted to using the
Agilent analyser, and am hoping I can make some sense of that data :)
cheers
Pow



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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