Very strange Northern problem

C. Griffin aniya at itsa.ucsf.edu
Tue Sep 23 16:42:16 EST 1997


Hello Mark,

If I fully catch your problem, I would suggest a few ideas. One regarding
your question as to your RNA samples running slow; possibly, I would
suggest trying a glyoxal gel system, it is easy, cheap, and I for one have
never had a migration problem using it. Formaldehyde gel on the other hand
have proven problematic and inconsistent. The glyoxal system is described
in Manniatis. 

If the gels suggest that the rRNA loads are equal, it is rather odd that
you have noticably different rRNA signal intensity between the two
samples, I guess in theory it could be a consequence of the transfer,
though I doubt it. 

Overall, the nonspecific hybridization to your rRNA, assuming that is what
is giving this signal, and the presence of smears in both samples may
suggest a problem with the probe. Try the following, 

1. Take one or more of your membranes which have been hybridized and
methylene-blue stain them; if the signal your seeing is rRNA the film
should line-up with the rRNA signal generated by the Met.Blue staining as
both are to scale. 

2. I don't know what type of probe you are using, but check the quality,
if it is in some manner degraded prior to use, it can result in a high
level of non-specific binding. 

I hope some of this proves relevant. 

Ciao,

Chandi Griffin









In article <mark_rose-2209971046400001 at 152.1.176.43>, mark_rose at ncsu.edu
(Mark S. Rose) wrote:

> 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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