TA cloning nightmare

[Bob Horton]

Catherine I. DiOrio wrote:
> 
> The polymerase you used to amplify your PCR product, KlenTaq, has
> 3'to 5' exonuclease activity...

KlenTaq is a truncated form of Taq, with the 5'-3' exonuclease domain removed. Taq 
doesn't have 3'-5' exo activity to start with, and none was added. It does have a lower 
mutation rate than regular Taq, but it is not due to any added exonuclease activity. 
Here's the ref:

Barnes WM. The fidelity of Taq polymerase catalyzing PCR is improved by an N-terminal 
deletion. Gene.  112(1):29-35, 1992 Mar 1.
   Abstract
     KlenTaq DNA polymerase is an N-terminally truncated Thermus aquaticus
    (Taq) DNA polymerase I. As expressed from a gene construct in Escherichia
    coli, translation initiates at Met236, bypassing the 5'----3' exonuclease
    domain of the DNA polymerase-encoding gene. A sensitive forward mutation
    assay was used to measure the relative number of mutations introduced into
    the entire lacZ gene by the polymerase chain reaction (PCR) under various
    conditions which allow the amplification of such a large DNA span. Two
    selectable markers, one at each end of the test lacZ fragment, were
    employed to avoid the plating and scoring of PCR artefacts such as primer
    initiation in the midst of the lacZ gene, and cloning artefacts such as
    empty vector plasmid. The measured relative mutation rate was twofold
    lower for KlenTaq as compared to the full-length Taq DNA polymerase.


---
Robert M. Horton
http://134.84.47.3                                       Have a :) day

"Scotty, try flushing the radioactive waste into the ventilation system!"