PCR/BglII digestion problems
bfrank at aardvark.ucs.uoknor.edu
Wed Mar 3 10:35:00 EST 1993
We have come across an aggravating problem recently and need some advive.
We used PCR to amplify a 600 bp region of the human genome. Through
sequencing portions of a human library, this PCR product should have 2
Bgl II site in it. They are not close to the ends of the PCR fragment
or close to each other. Our problems is that BglII does not digest
this DNA, but rather causes the DNA to migrate in a polyacrylamide gel
as a smeared "frown" slightly larger than 600 bp. The undigested &
untreated PCR product runs as a straight, horizontal band in these gels, as
does the PCR product following exposure to the restrcition enzyme buffer.
It is only after exposure to both this buffer and the enzyme that the
smeared artifact appears.
We have used other restriction enzymes to digest this product and they
work fine gicing the expected size bands in the gel. This includes the
use of Sau3AI which cuts within the expected Bgl II sites.
We have tried "cleaning" the DNA separately with phenol extractions, gene-
clean, and multiple ammonium acetate/ethanol precipitations, but none
of these removed the electrophoresis problem or the digestion problem. We
are in the process of sequencing through the putative BglII sites to be
sure they are present in the PCR product.
Can anyone suggest reasons for the unusual electrophoretic migration and
the inability of the enzyme to digest this sample?
Internet: BFRANK at AARDVARK.UCS.UOKNOR.EDU
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