digests direct in PCR buffer ?

Julian Dow GBAA02 at udcf.gla.ac.uk
Thu Nov 18 09:15:14 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



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