How do you determine % homology vs. stringency in a Southern Blot?

Charles A Miller oravaxcm at world.std.com
Sun Jun 2 23:02:32 EST 1996


I have been trying to determine how to calculate the Stringency of 
hybridization in a Southern Blot versus the % homology of the probe
with its various target sequences. For instance, from what I have been
able to determine, if you drop the stringency of a hybridization by 1 to
1.5 'C from the Tm of the probe, you also decrease the necessary homology
for hybridization by 1%. This is from Maniatis by the way. What
I cannot determine, is exactly at what point this applies, and
how exactly to determine the Tm using the equation (can't remember
it exactly but it looks something like this: Tm=81.5+16(log salt)-
molar content of G's+C's plus % formamide etc...). What I have to
wonder is this: When determining the Tm for the given conditions, do you
then have to determine the conditions separately for the hybridization as
well as the more stringent washes? The protocol I am using uses formamide
in the hybridization to lower the Tm, but the later washes do not use 
formamide. So, do I have to determine the Tm of the hybrid for this final
wash, subtract the ACTUAL temperature used in under the conditions
of salt and formamide plugged into the equation and then
use THAT to determine the % change in homology? ALSO, if I have 
digested the target DNA before I bind the probe, do I only
use the part of the probe that is actually binding to the 
fragment of most interest when I am trying to determine the proper
temperature? Do I also only use the number of basepairs that are binding 
to this particular fragment in the Tm equation (ignoring the rest of
the probe that will bind to another fragment that was cut away?

Is there a decent shareware/freeware mac or pc program for doing all
of this calculation?

Thanks!

Chuck Miller
oravaxcm at world.std.com




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