Trouble detecting prokaryotic mRNA

Karen L. Yoas kyoas at bcm.tmc.edu
Mon Jun 6 15:48:05 EST 1994


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.

Karen
   




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