PFGE/Southers

jdc6 at psu.edu jdc6 at psu.edu
Wed Feb 4 12:23:25 EST 1998


Dear Colleagues,

I am currently trying to map, via PFGE, the region around a given gene.
We have sequenced several thousand bp flanking this gene and it truns
out that these sequences are highly repetitive.

I have performed PFGE using a variety of restriction enzymes, followed
by Southern blotting using this gene, as well as two linked genes, as a
probe. In my Southerns, I am only getting hybridization to regions that
apparently represent repetitive regions in the genome (bands between
48-145 kb), as well as to the compression zone. In standard gel
electrophoresis, my probes work quite well. A very specific band with
little or no background hybridization.

I suspect (but I am not convinced) that the hybridization to the
repetive regions is non-specific. Is it possible that, using a variety
of enzymes, my probes consistently lights up repetitive regions, or is
this just non-specific hybridization. If so, does anyone have any
advice as to how I could improve the southern blotting.


Thanks in advance,
Joe Verica
Penn State
jdc6 at psu.edu



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