wgschech at med.uni-tuebingen.de
Fri Jun 26 04:49:04 EST 1998
I'd try one of the proofreading polymerases (Pwo, Pfu or anonther).
I'd purify the DraIII fragment and perform a 1 min extension reaction
as in PCR (72 degC) with all dNTP in the mix, for keeping the
equilibrium on the proper side. (If omitted, I'd expect an undefined
nibbling back activity of the Pol.
The crucial point should be that DraIII makes a 3' overhang (it cuts
GACNNN^GTG) and the proofreading Pol "sees" the upper strand being
to long. I wonder if this also would work with 5' overhangs, since in
this case, the shorter strand would act like a primer and the Pol
should make a fill in reaction.
Please correct me if I'm wrong.
Have a nice weekend,
> Greetings netters.
> I want to shave of a 3->5 cohesive end after DraIII digestion.
> Which idea seem better a T4 polymerase without nucleotides are using
> Pfu polymerase? Protocols for both would be most welcome. P.S I know
> fill-in would be simpler but for purpose it wont do.
usual disclaimers apply * This message is RNAse free - please don't touch!
University of Tuebingen, Germany
email: wgschech at med.uni-tuebingen.de * wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
More information about the Methods