David,
This has happened to me on occasion in the past. If you are using some
kind of commercial dot blotter to appply the DNA to your blotsand if you use
a different membrane for each organism one possible explanation (it
happened to me) is that you are not rinsing/washing the dot blot apparatus
well enough between organisms. Left over DNA from the previous sample will
transfer to your membrane from the o-ring or outer edge of the dotblot well
to your next sample. This would be the first thing that I look at.
Paul Vescio
Biology Deppt.
Rensselaer Polyechnic Institute
Troy NY
In Article <dsingle-171097090527 at bw-st1.mib.uga.edu>, dsingle at uga.cc.uga.edu
(David Singleton) wrote:
> 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 80oC 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 ug 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
