Burkhard.hassel at t-online.de
Sun Jul 16 06:22:46 EST 2000
On Sun, 16 Jul 2000 17:04:24 +0800, "Martin Chan"
<s987517 at mailserv.cuhk.edu.hk> wrote:
> 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.
your fear is unnecessary. At 37 degs the Taq-Pol is as active as a
stone. And trying to inactivate it by heat is a real bad idea. When
you run a PCR of, say, 32 cycles you have your 30 minutes already in
the 95 deg steps. And as you have bands ...
The best way of purifying the PCR product IMHO is to run 50% of the
reaction on a gel, excise the band(s) of interest and elute the DNA
using any standard starfleet protocol.
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