BGYCSW at leeds.ac.uk
Fri Dec 4 11:53:41 EST 1998
I have some discrepancies between restriction digest results
and sequence of PCR products. This concerns a HindIII site.
In some of my samples a HindIII site is not predicted and
this always matches with the digest, however in others a site
is predicted but occasionally this does not cut. There can
be, sometimes a partial digestion. It does not seem to be a
heteroplasmy problem since the automated sequence output does
not seem to be 2 peaks or even hint at this. I have tried 2
batches of enzyme. I have tried PEG precipitation of PCR
products to clean before cutting- this can help in inducing
partial digestion when previously there was none.
My questions are- is HindIII prone to not cutting certain
sequences for whatever reason? Is it perhaps "upset" by
residual PCR reaction components? (I have tried getting rid
of these with the PEG ppt). Has anyone any other ideas?
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