How much identity needed for hybridization?

Richard L lm11 at gpu.srv.ualberta.ca
Thu Nov 20 19:30:02 EST 1997


Hi,

Regarding how much identity are required for the hyridization, it's very
hard to say the consensus. As the case you gave, the probe is 500bp,
50%identity, if the identity is throughout the whole probe, it will be
very hard to find hybridization signal even you carefully wash or use
formamide. But if the identity is focused on a region of the probe, you
will find hybrization with careful washing. As for the background, from my
experiments, lower identity doesn't mean higher background as long as you
prehybridize it well.

Richard

"Christopher D. Skory" <SKORYCD at MAIL.NCAUR.USDA.GOV> wrote:
: Ignoring all of the mathematical equations for
: predicting Tm of probes (ie I don't want to hear about
: theoretical mol biol), what is the general consensus
: as to the minimum amount of identity needed for
: successful Southern hybridization?  Let's say the
: probe is at least  500 bp and random primed with
: either P32 or Dig.  Is it reasonable to find hybridization
: and wash conditions for target DNA with only 50%
: identity or would the background be too high?  Most of
: the probes that I have used have been at least 80%
: identity so I am very curious what experiences other
: people have had with heterologous probes.

: Christopher D. Skory, Ph.D.

: Fermentation Biochemistry Research
: National Center for Agricultural Utilization Research
: U.S.D.A.  Agricultural Research Service
: 1815 North University Street
: Peoria, Illinois 61604-3999

: E-mail:     skorycd at mail.ncaur.usda.gov




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