T-tailing for PCR product ligation

Duncan Clark Duncan at genesys.demon.co.uk
Tue Sep 13 03:47:30 EST 1994


In article: <350q8o$24d at styx.uwa.edu.au>  lsellner at uniwa.uwa.edu.au (Loryn Sellner) writes:
> 
> 
> 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)?

Cut your vector, phenol etc then put it in a Taq pol buffer and only 
add dTTP to 2mM plus say 5u taq pol per ug plasmid. Incubate for two hours
at 70C. Phenol etc then ligate. Run on LMP agarose and keep the 
unligated linear DNA band. This has the T on the end. Ligated DNA is 
either misligated or didn't add the T. About 50% will ligate. Use 
50ng DNA per TA cloning. Approx 50% should have your insert. Without 
prior ligation ratio is much worse.

Good luck

Duncan   

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Duncan Clark                        | Internet:    duncan at genesys.demon.co.uk
G4ELJ                               | Compuserve:  100015.1406 at compuserve.com
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