riboprobes for northern blots

Jeff Thornsberry jmt at biosci.mbp.missouri.edu
Thu May 25 12:51:47 EST 1995


In article <3psqrr$q9v at gazette.bcm.tmc.edu>, mg690769 at bcm.tmc.edu (Michael
Gerdes) wrote:

> Help!
> I am trying to use antisense riboprobes for northern blots, and have 
> come up with major backround problems- nomely hybridization to 28S RNA.
> 
> 
> general questions: did I use too much probe?
>                    were wash conditions addequate?
>                    any other helpful hints appreciated.

>                         mg690769 at mbcr.bcm.tmc.edu

Hi,
seems like you really do use too much probe. In a similar rxn i usually
use 0.03-0.1 ug
of template. Then, you washing might be pretty mild - but most impact
comes from 
the hybrn stringency. You may try this: hybridization - o/n at 55C in 50%
formamide,
 0.25M Na-phosphate (7ish), 0.25MNaCl, 2mM EDTA, 7% SDS + 20mkg/ml tRNA.
Wash in your solutions only, at 65C and longer 1st wash with stirring. With this
protocol i never failed (i use HybondN+ and 10mM NaOH-transfer) except a single
case when i got (don't laugh) a mighty signal corresponding to 28S rRNA band.
Good luck, Eugene



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