digesting PCR product with restriction enzymes (Question)

Rick L lm11 at gpu.srv.ualberta.ca
Fri Jun 26 15:04:31 EST 1998

Hi Eric,

It depends on what enzymes you want to use. for some enzymes, you can use
them directly(just take some PCR mix to a clean tube, add appropriate
buffer and enzymes), for example, BamHI, SacI etc. But most enzumes are
very sensitive to "whatever" in PCR mix, in this case you'd better remove

Usually I get rid of them by extracting with phenol/chl, 1x, and then ppt
with ethanol, and then dissolved in appropriate volume of ddH2O or TE

Another way is to run a aagrose gel, cut the band you want. I don't like
this way, because it needs 2 agarose gels - before and after digestion.

Hope this helps. If you have mre question, email me.


"Parent, Eric" <ParentE at dfo-mpo.gc.ca> 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.

: Thanks

: Eric

: ------------------------------------------------------------
: Eric Parent 
: Invertebrates and experimental
: biology division                              
: Fisheries and Oceans Canada	
: Maurice-Lamontagne Institute
: Mont-joli, Qc
: Canada

: parente at dfo-mpo.gc.ca
: http://www.qc.dfo-mpo.gc.ca/iml
: -------------------------------------------------------------

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