Q: Killing DpnI with heat?

Wolfgang Schechinger wgschech at med.uni-tuebingen.de
Fri Aug 1 08:32:22 EST 1997


Hi all Molbio Experts out here!

In a protocol for site directed mutagenesis 
(http://www.nwfsc.noaa.gov/protocols/pcr-mut.html)
the Dam methylated template plasmid is amplified with a Polymerase 
and then the parent DNA cut with DpnI.

In this protocol, the DpnI incubation is followed by a 30 min 72 degC 
step. As far as I know (the data sheet from NEB says) that DpnI can't 
be deactivated with heat. 
Then, the ligation of DNA follows.

So, folks, what is this step for? Since there still is polymerase 
(Pfu) in the cup, is it for proofreading? 

Any ideas, suggestions?


A nice weekend, 

Wolfgang
-----
                                     
Wolfgang Schechinger         
University of Tuebingen
email: wgschech at med.uni-tuebingen.de

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http://www.medizin.uni-tuebingen.de/~wgschech/research.htm




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