TA Cloning questions...

J. Hendriks hendrikb at worldonline.nl
Wed Aug 12 15:43:33 EST 1998


Koichi Kunitake wrote:
>   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>

We always use the TA cloning kit of Invitrogen. It's just a 5 minutes
ligation at room temperatuur and works perfectly. They don's use T4 DNA
ligase, but use topoisomerase instead. Perhaps you can try this one.
Some time ago I did ligations overnight at 16 degrees and over weekend at 4
degrees. I don't know why the protocol of Promega is saying 4 degrees.
Hope this is of some help
Brenda Hendriks
b.hendriks at wkz.azu.nl
hendrikb at worldonline.nl






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