Hi Kunal,

I have done some post PCR ligation experiment before. In order to insure that there is 3'-A overhanging(I believe that's what you want as well) , we always include a 3'-A tailing step which incubate dATP, DNA and polymerase for an extra time depending on the polymerase you use. 

Hope that helped you.

High fidelity polymerases have proofreading activity as part of the polymerase, that is what makes them high fidelity. Taq polymerase does not. It is the proofreading activity that removes the overhang. 

If you want to use a high fidelity polymerase, you won't get a 3' overhang because high fidelity polymerases contain 3' to 5' exonuclease activity. However, you can always design your primers to add a restriction site and a little extra junk DNA on the end of the sequence so that you can perform a restriction digest on your PCR sample to prepare it for a ligation reaction.

Hi there,

If you want to introduce 3'A to your PCR product you would have to treat it with Taq polymerase in presence of dATP as follow:

http://www.openwetware.org/wiki/Addition_of_3%27_A_overhangs_to_PCR_products

Thank you very much, for all your responses!