digests direct in PCR buffer ?

txpljfg at UABCVSR.CVSR.UAB.EDU txpljfg at UABCVSR.CVSR.UAB.EDU
Fri Nov 19 14:44:51 EST 1993


> I thought the idea in the title was great. I remember the thread from a few
> weeks ago, and I'm trying an Eco/Bam double digest this minute. I noticed
> that Taq buffer has one tenth the Mg, and one half the salt, of React 3, so
> I analysed 5 ul of a 20 ul reaction on a gel, diluted the rest to 50 ul
> with 1xReact 3, then added 5 U of each of eco and bam. 
>  My big question, with more general relevance, is why doesn't Taq pol fill
> in the recessed ends generated by the enzymes? Is it assumed to be dead?
> Does it have negligible activity at 37C? Are the nucleotides depleted? Is a
> heat inactivation step compulsory? How *do* you heat inactivate Taq pol? 
>  Presumably, if the Taq fills in a sticky end, it is no longer recleavable,
> so I opted for an excess of restriction enzyme and a short time. Does
> anyone have an answer to this, or happen to know the temperature dependence
> of taq pol?
> 
> -- 
>  - Julian Dow
> 
The activity of Taq pol will depend on how many cycles you have run: 
as a general rule of thumb, about half the activity of taq pol is gone
after 40-50 cycles.  Also at temperatures above 94 degrees, the
half-life of taq drops off very rapidly.  We routinely heat-inactivate
taq pol by heating to 99 degrees for 10 minutes.  While I have never
actually tested the activity of the enzyme after such treatment, I have
encountered no problems.

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James F. George, Ph.D.              "Science is simply common sense at its
Department of Surgery                best -- that is, rigidly accurate in
University of Alabama at Birmingham  observation, and merciless to fallacy
205-934-4261 voice                   in logic"   --Thomas Huxley
txpljfg at uabcvsr.cvsr.uab.edu
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