Dot-blot Hybridization Question

PAVS Paul A. Vescio vescip at rpi.edu
Fri Oct 17 08:26:46 EST 1997


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



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