Cutting a PCR-product

Dom Spinella dspinella at
Wed Aug 12 10:04:05 EST 1998

> Hi,
> I want to confirm the sequence of my PCR-product with a digestion. Can I
> perform the
> reaction directly, only adding RE and the buffer for RE or do I have to
> extract the DNA from the PCR-mix.
> Thanks in advance,
> Chris

It may depend some on the restriction enzyme, but yes -- you can often
directly digest the PCR product as you suggest, although I have found
that you have to hit it pretty hard (up the RE units by at least a
factor of 5).  I published a paper a several years ago describing the
technique (Spinella, D.G., 1990, "A rapid method for genotyping mice for
T-cell receptor VBa and VBb haplotypes by PCR analysis of whole blood"
J. Immunol. Meth. 135: 209-212).  I'll send you a reprint if you like.
Cheers. --Dom Spinella

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