Very strange Northern problem

Mark S. Rose mark_rose at ncsu.edu
Mon Sep 22 05:46:40 EST 1997


Hi folks

   I am having a very strange problem with my northern blots.  I am
probing total RNA from a strain with a disrupted copy of my gene of
interest and a strain containing multiple copies of the intact gene.  In
my earlier northerns the multicopy strain produced a very intense signal
while the disruptant did not.  The problem is that now neither strain
produces a signal corresponding to the transcript of this gene (based on
the physiology of the strains used for these RNA preps I am confident that
abundant transcript should be present in the multicopy strain).  What I do
see is: 1) hybridization to the rRNA bands that is stronger in the lane of
the multicopy strain; 2) hybridization to a high molecular weight compact
smear (diffuse band) that is very strong in the multicopy strain but
almost absent in the disruptant (at present I hypothesize that this signal
is due to contaminating genomic DNA); and 3) high to low molecular weight
smears in both lanes but stronger in the lane of the multicopy strain.
I've tried making new blots and using different preps but I get the same
result.  When I use my probe against a Southern of genomic DNA from the
wild type the pattern is exactly as it should be.  The formaldehyde gels
used to make the northerns have compact rRNA bands and both lanes appear
equally loaded.  The genomic DNA contamination of my samples (you can see
it on TBE gels though not the formaldehyde gels) may in part explain why
the prep from the multicopy strain gives a generally more intense signal
than the disruptant prep but this doesn't seem to explain the absence of
hybridzation to the gene's transcript.  FYI I am blotting to NYTRAN PLUS
using 20X SSC.  To fix the RNA I have either UV crosslinked my filters or
baked and then crosslinked them.  Could somthing be wrong with my samples
such that the mRNA runs anomalously slow thus providing an alternate
explanation of observation #2?  I would appreciate any suggestions.

Thanks 
Mark



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