I am trying to clone a PCR product that was amplified using Pfx polymerase into pGemT vector. I had to A-tail the PCR product using Taq polymerase since Pfx only generates blunt end products. My ligation reaction was successful, however when I got the sequences, there is an extra T between the 3'end-T of pGem and the start codon of my product. It only happens in the 3' end of pGemT (or 5' end of my product). Can Pfx polymerase generates PCR products with just one 3'end overhang? or Can Taq polymerase add two consecutive dATP during the A-tailing?
Please write me your thoughts ASAP, it has happened twice and it's a pain already :(
GGC CGC GGG A-T-T-T-G ATG GGA AGC ATG AAG
The two Ts in Bold belong to pGemT, the T Italic is the extra T I've got from the sequencing reaction. After the T in Italic you may find the 5'end of my insert. The second T (in bold) from pGemT is the one that ligates with the overhang A added through the A-tailing protocol.