Problems with restriction digests
hopkinsc at lincoln.ac.nz
Fri Jan 23 16:01:18 EST 1998
We are having problems with our restriction digests that I am hoping someone
will be able to solve.
We are digesting PCR products that have not been cleaned up prior to digestion
and are using 6 U of enzyme for 100-200ng of PCR product in an 18 ul reaction.
Digestion is carried out for 2 hrs with optimal buffer and temperature.
Our problem is we aren't sure if the products are cutting to completion, and
we aren't sure how to determine this or how to improve the digest to get it to
We have two species one which gives a band at 900 bp after digestion and the
other which gives two bands 650 and 250. However sometimes this second
species will also have a faint band at 900 which we presume is incomplete
digestion and we ignore. What we are concerned about is how can we be sure
that the first species doesn't have the restriction site to cut it into 2
bands? Is there anything that could be inhibiting the digestion? Would
increased enzyme/time/buffer/whatever cause the band to cut. Is there
anything you can add to the digest to improve it (like you can add 5% DMSO to
the PCR)? We did wonder about BSA, but most buffers if they need it all ready
have it. Both species are about the same concentration in the digest.
Hope someone can help
Charlotte Cameron email: hopkinsc at lincoln.ac.nz
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