bhjelle at vaxine.unm.edu
Thu Jun 25 10:41:04 EST 1992
In article <1992Jun25.113339.65 at chmeds.ac.nz> cytogen at chmeds.ac.nz writes:
>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.
>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.
Thanks, Martin. For the curious, I am now trying the 2nd method
(pg 1156), involving TdT and ddT tailing. Got *lots* of colonies :-).
If you don't hear back from me, that means they were duds :-(.
More information about the Methods