Is A-tailing of restriction fragment possible? - (Mar/02/2006 )

I wonder if it´s possible to digest DNA with a blunt cutter, purify it on a gel and a-tail it with Taq and dATP afterwards in order to clone it into a pGEM-T cloning vector. Until now I´ve only heard of A-tailing PCR-fragments. Does it make a difference?

The aim is to subclone a large fragment (10kb or so), blunt end ligation didn´t work so far, so I hope the A/T-method would improve things.

Thank you for your help.

no problem
we even using dNTP in stead of dATP during incubation with Taq, so it will not limited to blunt end fragment..
In fact you might add A before purification from gel, surely you should purify digestion (phenol/chloroform extract and ethenol precipitate) before incubate with Taq and dATP/dNTP
hope it work for you

Thank you, rshi, for your prompt answer.

I´ll give it a try today. Keep your fingers crossed.