PCR/BglII digestion problems

Andre Hamel hamel at ccu.umanitoba.ca
Wed Mar 3 16:28:38 EST 1993


In article <3MAR199309352672 at aardvark.ucs.uoknor.edu> bfrank at aardvark.ucs.uoknor.edu (FRANK,BART) writes:
>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?
>
>Thanks,
>Bart Frank
>Internet: BFRANK at AARDVARK.UCS.UOKNOR.EDU


Have you tested your Bgl II on other DNA such as plasmid vector in order
to ascertain if the enzyme may be contaminated with any other nucleases?

Andre Hamel
Manitoba Vet. Virol.
Winnipeg, MAnitoba
CANADA

email: hamel at ccu.umanitoba.ca



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