Dig labeled probes

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Wed Sep 14 10:42:22 EST 1994


4700gbera at umbsky.cc.umb.edu wrote:

> The only time I've ever had problem reusing a dig-labeled probe was when
> I didn't boil it long enough to fully denature it. Of course, one of my probes
> is 16 kb, and it's in about 70 mls. I usually  boil it for about 1/2
> hour to convince myself that it's fully denatured. <snip>

DNA denatures immediately upon exceeding its Tm.  So if you want to 
convince yourself that it's denatured, use a thermometer.
The length is irrelevant to denaturation.
Longer DNA will reanneal faster, but since most labelling procedures
fragment the probe, this will also be irrelevant.
With a large probe volume, you might worry that you'll lose probe
to reannealing because you can't quench it as fast.  However, at
the usual low probe concentrations, the amount of reannealing shouldn't
become a factor unless you leave it at a reannealing temp. a very long
time before getting the filter in.

Steve Hardies., Dept. of Biochemistry, Univ. of Texas HSC at San Antonio
Hardies at thorin.uthscsa.edu




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