RNA denaturing gels

[IanMc]

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