unspecific (was: RNA stability) for Susanne!!!

Mart Speek smart at KBFI.EE
Tue Nov 23 05:05:49 EST 1999


Dear Susanne:
\your reply address turned out to be incorrect one,
(srohrerNOsrSPAM at immv.unizh.ch.invalid)\

As you mention ... almost
EVERYONE has the problem of unspecific hybridization to the rRNA bands.

Regarding the hybridization of 28S rRNA, but not 18S rRNA,
I seem to remember that when using riboprobes derived from
a Bluescript or similar vectors, multicloning site sequence included
in the probe has 7-9 bases matching regions in 28S rRNA;
also remeber that rRNA is in a huge excess (~70% of total RNA)
that's why even a small region identity but presented abundantly
can drive equilibrium toward unwanted hyb.

Likewise, 23S rRNA (and perhaps 16S rRNA) may be capable of yielding
a hyb-positive band, however its image should be recognized
(usually large volume) from the blot.

It is difficult to get rid of this unwanted hyb. From my experience,
I would recommend to increase the hyb temp ~80degr Celc;
and wash at high stringency
(nonformamide cocktail, preferably made in 7% SDS)

Hope this helps.

Mart (ESTONIA)







More information about the Methods mailing list