TA cloning

Bryan L. Ford fordb at bcc.orst.edu
Sun Jun 21 22:37:44 EST 1998


Wolfgang Schechinger wrote:
> 
> Does this mean that Taq adds a dT under these conditions?
> Will this work with Pwo or Pfu, too? (This would be the better
> enzymes for cloning IMHO, since they have a lower error rate)
> 


Yes, as I understand it Taq merely has some preference for "A-extendase"
activity, and that it will add any other base if given only that
particular dNTP to work with. Extendase activity is definitely not seen
with straight Pwo or Pfu. The lower error rate of Pfu is directly
related to its 3' to 5' exonuclease activity. Boehringer's "Expand High
Fidelity" uses a mix of Taq and a bit of Pwo. The error rate is quite
low but there are enough A's overhanging that T-A cloning also works.
This may fit your expectation of the the best of both somewhat
incompatible situations.

-Bryan



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