28S doesn't blot!

Stefi~ gifalone at tin.it
Wed Jun 5 14:04:46 EST 2002


I use a 50mL gel with 2,5ug EtBr (approx height: 6mm) and my RNA is from
Bufo bufo. I B.bufo's 28S doesn't have a nick because I've been making RNA
denaturing gel electrophoresis analysis with that species for almost 2 years
and 2 clear bands have always appeared.
I'll try less EtBr and a thiner gel.
Thanx a lot for all your suggests.

Stefano Falone
xxxgifalone at tin.it (rimuovere xxx)
http://web.tiscalinet.it/gifalone
ICQ 110419911
"Nick Theodorakis" <nicholas_theodorakis at urmc.rochester.edu> ha scritto nel
messaggio news:3cfd6898.49632904 at netnews.worldnet.att.net...
> On Tue, 04 Jun 2002 19:21:35 GMT, "Stefi~" <gifalone at tin.it> wrote:
>
> >Hello.
> >How may I solve a blotting issue during my Northern?
> >After a 12h transferring, if I put my nylon + membrane on a UV light
system
> >I cannot see 28S band, while 18S blots regularly.
> >I use EtBr for gel (1,3% agarose) staining, 10X SSC and 1kg weight in
> >blotting apparatus.
> >Please, help me.
> >Thanx in advance.
> >
>
>
> Can you see it on the gel before you blot? If so, is it still in the
> gel after you blot?
>
> Maybe you have too much weight, and the gel is getting squashed too
> much, which would inhibit the transfer of high mol. wt bands. You
> could also try a brief base hydrolysis to nick the large RNAs before
> transfer. Incubate with 50 mM NaOH for about 20 min, then with 0.1M
> Tris, pH7.4 for another 20-30 min. Then use about half the weight you
> are using now. And also maybe use a 1.2% or less gel.
>
> Nick
>
> --
> Nick Theodorakis
> nicholas_theodorakis at urmc.rochester.edu


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