digesting PCR product with restriction enzymes (Question)
mwcrepeau at ucdavis.edu
Fri Jun 26 17:48:50 EST 1998
It varies widely from enzyme to enzyme. But for many of them, the
answer is yes, you can simply add the enzyme directly to the completed PCR
reaction. This is particularly true if you are in the habit of digesting
with one microliter of enzyme. Since most enzymes are sold at 10
units/microliter you'll have more than enough activity to digest an
average PCR even if the enzyme isn't particularly happy in the PCR buffer.
We routinely digest unpurified PCR reactions in our lab. In fact it is
funny because one undergraduate who had been working for months in the lab
running and digesting PCRs for RFLP analysis asked me one day "why do they
always send these little tubes of buffer with the restriction enzymes?"
She'd never used them and didn't know what they were for!
If you are unsure about a particular enzyme, you can simply test it
empirically to compare its performance in your PCR buffer with its
performance in the manufacturer's buffer. Also, if you can get your hands
on a copy of Biochemica No. 2 (it's a trade publication by Boehringer
Mannheim I think) there is a good write-up on this subject on page 14. It
includes a table that lists the activities of many common enzymes in PCR
buffer relative to their activities in the manufacturer's buffers.
<mwcrepeau at ucdavis.edu>
In article <3B675D1AC3E1D111BC1600805FEACC6701FB57 at lauimls05.qc.dfo.ca>,
ParentE at dfo-mpo.gc.ca ("Parent, Eric") wrote:
> Hello all.
> When digesting PCR product with restriction enzymes. Does the Taq, the oil
> or whatever in the PCR inhibits the action of the restriction enzymes. My
> question is: cant you use restriction enzymes directly on the PCR products
> in question or do you have to get rid of all the "junk" first.
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