Km of rest. enzymes.

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Thu Sep 29 14:25:48 EST 1994


John H. McDonald writes:

> Does anyone know the Km for DNA of a "typical" restriction enzyme (or of
> AccI in particular)?  I'd like to digest a rather small amount of DNA, and
> I'm wondering if I need to add some other, generic DNA in order for the
> enzyme to work.  I'm hoping to digest one of two alleles in a heterozygous
> individual, then PCR amplify the uncut allele. 

The Km won't help you.  The problem with putting a tiny amount of DNA in
with the r. enz. is that the small amount of contaminating general nucleases
that come in all these commercial preps will destroy your DNA.  So you
shouldn't work with less DNA that would be a 100 x overdigestion.  The
problem with diluting the enzyme down proportionately is that enzymes
are subject to denaturation in dilute solution.  You can try to stabilize
the enzyme with gelatin or BSA (I prefer gelatin because you can autoclave
it) and/or keep the digestion time real short.
However, I'd recommend adding the carrier DNA and using more standard
r. rxn. conditions if the carrier DNA doesn't interfere with other aspects
of your experiment.  This way your DNA is also protected from getting lost
during pipetting, etc.  There are some other things that can be used as
carrier for the DNA if necessary.

I'm not sure I understand your experiment, but wouldn't you be happier
to amplify first and then cut?  The way you're proposing to do this,
I don't know how you'd ever be sure you actually cut to completion.

Hope this helps,

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


 




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