28S doesn't blot!

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Tue Jun 4 20:27:59 EST 2002

On Tue, 04 Jun 2002 19:21:35 GMT, "Stefi~" <gifalone at tin.it> wrote:

>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 Theodorakis
nicholas_theodorakis at urmc.rochester.edu

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