Trouble detecting prokaryotic mRNA

Cornelius Krasel krasel at alf.biochem.mpg.de
Tue Jun 7 07:35:19 EST 1994


Karen L. Yoas (kyoas at bcm.tmc.edu) wrote:
: I am having trouble detecting mRNA from E. coli.  The cells were taken
: mid-phase and RNA extracted w/ RNAzol B procedure.  I have lots of RNA
: (intact).  I use formaldehyde gels with MOPS buffer.  I have tried
: capillary transfer and electroblotting with no success.  Hybridization is
: done w/ SSC, SDS and Blotto (powdered milk).  Prehyb is done for 1 hour at 65C
: and hyb goes ON at same temp.  Washes are SSC/SDS and SSC at 65C and RT, resp.
: I have tried the Genius System using the colometric detection as well but
: with no luck.  My oligo probe is 1 kb and has been tried on a southern blot to
: dbck it.  The amt of RNA in ea. well has been from 10-30 micrograms.  I
: have been told to use all of my RNA sample (300 micrograms) on the next
: gel.  Won't this cause some distortion problems with so much loaded?  If
: anyone has any ideas please contact me.  I have worked with eukaryotic RNA
: before but this is the first time with prokaryotic RNA.

In my experience only _very_ abundant RNAs can be detected in E. coli by
Northern blotting (for example, Northern blotting worked in my hands for
ompA mRNA but not for ompC mRNA). Maybe you have to resort to something
more sensitive (e.g. primer extension).

(I assume that you can see the transferred RNA on your blot by staining
 with methylene blue.)

Hope that helps,
--Cornelius.

--
/* Cornelius Krasel, Abt. Lohse, Genzentrum, D-82152 Martinsried, Germany */
/* email: krasel at alf.biochem.mpg.de                 fax: +49 89 8578 3795 */
/* "People are DNA's way of making more DNA." (Edward O. Wilson, 1975)    */



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