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Best Taq for colony PCR? - (Aug/14/2014 )

I was hoping someone here might have experience of multiple options for colony PCR. NEB recommends their OneTaq and Promega recommends their GoTaq. I'm guessing there are many others from different manufacturers. Has anyone any experience to suggest that one is better than another for this purpose?

 

The OneTaq protocol accepts bacteria directly in the reaction buffer while the GoTaq needs the bacteria to be boiled and centrifuged first which is worth knowing.

-seanspotatobusiness-

I've never had any problems with standard Taq from a number of suppliers.

-bob1-

The boil/spindown protocol is a bit more conservative, but its use is almost certainly independent of the brand of Taq that is used. Pretty much Taq is Taq is Taq.

-phage434-

We are using hot-start Taq version, that requires 15 minutes initial denaturation at 95.

That essencially (apart from it's hot start function) "boils" anything that is in the reaction, bacteria included.

-Trof-

We are using hot-start Taq version, that requires 15 minutes initial denaturation at 95.

That essencially (apart from it's hot start function) "boils" anything that is in the reaction, bacteria included.

 

Interesting;  I occassionally use Promega's HotStart G2 Master Mix.  It's complexed with an antibody which required a denaturing at 95C for 2 minutes.  Did you extend the recommended incubation time at 95 C, or was that what was recommended by the manufacturer?

-djvan-

No, that would damage polymerase, we use QIAGEN HotStarTaq, which is covalently blocked (not by antibody) and 15 minutes is standard recommended initial denaturation.

But there have to be others that also have longer denaturation times. Usually 5 minutes is what I met among the antibody-blocked HS polymerases, but the one in our real-time mastermix can be activated by 5-10 minutes.

-Trof-