Taq inactivation

tfitzwater at gilead.com tfitzwater at gilead.com
Mon Jul 17 12:39:06 EST 2000


Subject: Re: Taq inactivation
From: Arnoud van Vliet (avvliet at knoware.nl)
Date: Sun 16 Jul 2000 - 10:45:38 BST

> I am going to directly use the PCR mixture for restriction digestion,
> but I fear the remaining Taq in the PCR mixture will fill in the 5'
overhang
> generated by restriction endonucleases. Is my worry necessary? Can Taq be
> inactivated by heating at 94-96C for 30 min prior to restriction
digestion?
>  I am frustrated by the low recovery of my lab's PCR purification kit, so
I
> plan to skip the purification step.

According  to the literature, Taq can survive phenol:chloroform extraction,
ethanol precipitation, gel electrophoresis, column purification and can add
extra  nucleotides to PCR-generated inserts whether they are blunt-ended or
post-restriction  digestion  products, resulting in low efficiency cloning.
Wayne  M.  Barnes 1992 Gene 112, 29-35.  Jiang, G., L. Nepomuceno and F. M.
Sladek, 1995.  Exclusive homodimerization of the orphan receptor hepatocyte
nuclear  factor  4  defines a new subclass of nuclear receptors.  Mol. Cell
Biol.  15:   5131-5143.   Sanhau, G. S., J.W. Precup and B. C. Kline, 1989.
Rapid  one-step  characterization of recombinant vectors by direct analysis
of   transformed   Escherichia  coli  colonies.   BioTechn.   7:   689-690.
Wolfgang  A.  Wybranietz  and  Ulrich Lauer, 1998.  Distinct combination of
purification  methods  dramatically improves cohesive-end subcloning of PCR
products.  BioTechn. 24(4):  578-580.

Tim Fitzwater
Principal Research Associate
Gilead Sciences


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