TA Cloning questions...

Koichi Kunitake kunitake at salk.edu
Sun Aug 9 22:00:43 EST 1998


   I was reading the Promega TA cloning guide to its pGEM system, and it says:

Low temperature ligations (i.e., 4oC) favor annealing of the A-overhang of
the insert with the T-overhang of the vector. Higher temperatures (i.e.,
room temperature) give rise to a high background of blue colonies
resulting from optimization of blunt-end ligations of non-T-tailed vector.

Should T-A ligations be done at 4C? I always thought that they should be
done at 15-16C. 

Also, I was wondering it it would be better to use E Coli DNA ligase
instead of T4 DNA ligase in the ligation, since the E Coli DNA ligase
would be unable to close up the blunt ends, but hopefully would be able to
covalently join T/A overhangs. (1 base counts as "cohesive", right?) Has
anyone tried this?

I also read that adding PNK and 1mM ATP to the ligation reaction helps in
the final ligation. Is this the case?

Thanks for reading this,

Koichi Kunitake
<kunitake at salk.edu>



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