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 :(


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.

  • 1
    $\begingroup$ Pfx wont add an overhang. Check this. Taq generally adds one A $\endgroup$ – WYSIWYG Jul 9 '13 at 20:46
  • $\begingroup$ Could you post sequences at the two junctions, just to make the problem absolutely clear? $\endgroup$ – Alan Boyd Jul 10 '13 at 6:51
  • $\begingroup$ Hi, Thanks for your answer! I have included above the sequences at the two junctions as you requested. Hope this helps to better understand the issue. $\endgroup$ – pepe84 Jul 10 '13 at 12:42
  • $\begingroup$ did you check the electrophretogram of your sequencing reaction. just to make sure that it is not an erroneous base call $\endgroup$ – WYSIWYG Jul 10 '13 at 13:02
  • $\begingroup$ Yes, I've checked every peak of the electropherograms and there is clearly an extra T there. From four clones I've sent for sequencing, all of them had this extra T. I have called promega to confirm it is not a vector issue. They said it is possible that they may have produced a wrong batch of pGemT with an extra T. I can't believe that was the case though. Anyways they will possibly send me a new batch next week. If you think that might be a vector issue, please let me know. $\endgroup$ – pepe84 Jul 10 '13 at 14:14

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Browse other questions tagged or ask your own question.