Unusual background problems on Southern blots
steffen at hawk.bcm.tmc.edu
Fri Aug 10 09:44:50 EST 1990
We have recently been having a problem with our Southern blots which in some
ways acts like repetitive sequences in the affected probes, but in other ways
doesn't. I wonder if anyone else reading this group has experienced similar
problems, and what they did about it.
We have isolated a series of clones from a region of the rat genome (Dsi-1).
All of these clones have been screened for rat repetitive sequences by
hybridization of the clone DNAs on a filter to radiolabled rat DNA, and by this
criterion have been found to be free of repeats. When we then use the clones
as probes to screen DNA from a variety of species (rat, mouse, hamster, rabbit,
human) we find that some of the clones produce nice blots as expected, but that
two of the probes produce a high background reminiscent of repeats. What is
odd is that the background is NOT seen in rat DNA, and is seen in only SOME of
the other species. My previous experience with repeats in probes is that
background is only a problem with DNA of the same species as the probe was
derived from (resulting from rapid evolution of repetitive sequences). Even
given an evolutionarily conserved repeat, why should it be present in only one
copy in the species of isolation (rat) but in many, many copies (producing a
smear on the blot) in some other species (mouse, hamster)? Not suprisingly, we
are considering the possibility that this is some kind of technical artifact,
but we haven't been able to think of what kind. Any suggestions along those
lines would be welcomed as well.
If you have any insight, please respond to me by email, and I will post a
summary of any responses to this newsgroup. Even if I receive no response, I
will summarize that fact (after about two weeks). Obviously, responses
directly to this newsgroup are infinitely preferable to no response.
Thanks in advance!
-David Steffen- (steffen at mbir.bcm.tmc.edu)
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