TA Cloning

cytogen at chmeds.ac.nz cytogen at chmeds.ac.nz
Wed Jun 24 17:33:38 EST 1992


In article <d3llx7c at lynx.unm.edu>, bhjelle at vaxine.unm.edu (Brian Hjelle) writes:
> Both my lab and an adjacent lab had spectacular flops with TA cloning
> recently. We had PCR product we assumed to have an additional A
> on each 3' end, and cut pUC or GEM with HincII, followed by incubation
> for 1-3h at 65-70C with 1mM dTTP and 5U Taq polymerase per ug of
> plasmid. We each got colonies but they were just pUC or GEM.
> 
> I have been unable to find the NAR article describing how this
> should be done. Does anyone have the reference?  Or any suggestions?
> 
> Thanks,
> Brian


There are at least two ways of making "TA" cloning vectors: one with addit
ion of ddT by terminal transferase, the other by incubation of the vector
with Taq in the presence of dTTP.  The references are both in NAR 19(5), 
page numbers hard to read, but maybe about 1154!  Both methods work, but
in my hands not very well - results are variable, for different vector
preps and different inserts.  I don't get much better efficiencies this
way than if  I just polish the insert ends with T4 pol and clone into
dephosphorylated, blunt cut vector, or just kinase the insert and clone
into blunt, undephosphorylated vector and select with blue/white or
colony lifts.  I still find incorporating linkers in the primers and
using these for sticky-end cloning is the best and most reliable method.

Cheers,

Martin



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