digesting PCR product with restriction enzymes (Question)

Wolfgang Schechinger wgschech at med.uni-tuebingen.de
Fri Jun 26 12:52:19 EST 1998


Hi Eric!

Normally, I separate the PCR sample on an agarose gel and isolate 
my band of interest to prevent template material from being cloned.
Another approach could be (if you re-amplify a band from a methylated 
plasmid) adding DpnI and Buffer to the PCRmix, digesting the template 
into pieces and then cut the amplified fragment with other enzymes. 
Or vice versa, depends on the buffer the restriction enzymes need. 
Then you can transform this digestion mix directly.

If you just want to test a PCR band's identity, you simply can add 
the enzyme to the PCR mix. normally, Taq or other compounds shouldn't 
harm the digests, if the salt concentrations and the pH are 
compatible, at least to my experience.

Wolfgang

> 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
> -------------------------------------------------------------
> 
> 
> 
-----
usual disclaimers apply * This message is RNAse free - please don't touch!
-----                              
Wolfgang Schechinger         
University of Tuebingen, Germany
email: wgschech at med.uni-tuebingen.de * wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
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