Killing Taq

Warren Gallin wgallin at gpu.srv.ualberta.ca
Mon Oct 2 19:44:22 EST 1995


In Article <44pfm3$fv at cerberus-138.wustl.edu>, HAVILAND at KIDS.WUSTL.EDU
(David L. Haviland, Ph.D.) wrote:
>In <DFKoHC.851 at news.cis.umn.edu> horton at biosci.cbs.umn.edu writes:
>
>> jim hartley (jhartley at access2.digex.net) wrote:
>> : Has anyone tried inactivating Taq polymerase (so restriction enzyme ends 
>> : are not blunted) by zapping the dNTPs with alkaline phosphatase? Protocol 
>> : would be easy: add CIAP, incubate, kill with 94 C incubation, then 
>> : ethanol precipitate and cut with RE.  Seems like it should work.
>> 
>> You CAN kill Taq with heat, too; how 'bout a 99 C incubation for 20 min?
>
>Hi:
>
>A long incubation >95'C *should* do the trick.  But that would depend on 
>the particular enzyme.  If I recall, Hot-Tub had a longer life at >95'C 
>than did native Taq - but then it has been about 4 years since I've seen 
>the activity profiles.  However, in his making and purifying KlenTaq, Wayne
>Barnes described his findings in his Gene 112:29-35 (1992) paper.  It is
>buried in the fine print of the Mat & Methods.  There, he found that Taq
>survived phenol, chloroform, and EtOh precipitation by demonstrating
>32P-dNTP end filling activity by Taq.  The only way he found to completely
>inactivate Taq (KlenTaq) was by a PEG precipitation of the final PCR
>product. 

In addition, after 20' at 99 deg C, your DNA will all be single stranded,
and you will have to be reasonably careful in trying to anneal it back to
double stranded.  I think PrK digestion is a better solution, both in theory
and in practice.

Warren Gallin,
Department of Biological Sciences, University of Alberta
wgallin at gpu.srv.ualberta.ca



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