Q: Killing DpnI with heat?
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
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,
University of Tuebingen
email: wgschech at med.uni-tuebingen.de
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