RNA denaturing gels

corona via methods%40net.bio.net (by customer from newsnet.com)
Tue Sep 25 03:37:07 EST 2007


>> > 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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