I am currently beginning to work with dot-blot hybridizations, and
while
recently running some standard controls I found some unexpected results.
The membrane is Boer-Man positively charged nylon. I am binding
genomic
DNA from _E.coli_, Yeast, and _Methanococcus maripaludis_ (Archaea) to the
membrane using baking at 80¡C for 2 hours. The probes are 5'-end labeled
with 32P-ATP using T4 PNK. The probes themselves are domain specific for
Universal, Eucarya, Bacteria, and Archaeal sequences in the 16S rDNA.
In performing the hybridization (and subsequent exposure to a
phosphorimager
plate [Molecular Dynamics]) with the labeled Universal probe, the expected
results were obtained; namely, a linear graph of µg DNA spotted vs
"counts". The membrane was stripped of probe using a NaOH treatment and
neutralized with TE buffer (pH 7.5).
The same membrane was then hybridized with the Bacterial-specific
probe.
As expected, the E.coli showed a linear pattern of binding, the Yeast
showed some low binding (presumably due to the mitochondrial DNA),
however the spotted archaeal sequences produced some strange results.
Rather than no to little binding as expected, there were "halos" around
the dots; high circles of activity around the edges of the dot with areas
of low binding in the center. This did not happen anywhere else on the
membrane. Analyses of the "halos" showed that the probe did not bind in a
linear manner. Exposure of the membrane between hybridizations showed most
of the universal probe stripped, so that probably wasn't the problem.
Background is low in all exposures.
Does anyone with more experience in this procedure have any suggestions
as to what could results in "halos" around a dot in a dot-blot?
Thanks in advance.
David Singleton
Department of Microbiology
University of Georgia
