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
them. 

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
buffer.

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.

Rick


"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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