T-tailing for PCR product ligation

[J Shepherd]

szcooley at chip.ucdavis.edu () writes:

>G Selvarani (medp3056 at leonis.nus.sg) wrote:
>: Loryn Sellner (lsellner at uniwa.uwa.edu.au) wrote:

>: : Dear netters,
>: : I have been reading the advice for cloning of PCR products by using the 
>: : TA cloning vector (or any T tailed vector). Is there a way to T tail my 
>: : own vector so I can clone my PCR product into the site I want, (namely a 
>: : blunt ended restriction site)?
>: : Thanks.
>: : Loryn

>: dear loryn, 
>: you can prepare your own ta vector using preferably a vector containing 
>: Eco RV site.after the blunt ending,incubate the restricted dna with Taq 
>: polymerase and dTTP at 72oC for about 2 hours and clean them as normal dna.
>: there was an article regarding this procedure in Nature of either May,Jun 
>: or July 1994.If you can't locate it E-mail me and i'lltry to search for you.
>: I've tried it and it works as good as the commercial ones.
>: Happy trying!


>: arun


>I have tried this with Bluescript vectors and it didn't seem to work. Is 
>one TAQ polymerase as good as another for this operation?

	Not all polymerases appear to enable t-tailing, regardless of
the A-overhang. Also, we found the ligation conditions to be much more
critical than for say, blunt ended. But when they were right... :-)

	Jason Shepherd. ICMB, Edinburgh.

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