Cloning problem into pGEMT-easy - (Jul/22/2012 )

Hi all,

I am having trouble transforming a PCR product into PGEMT-easy vector. I have performed overlap extension PCR, followed by fusion PCR. Both the PCR's have worked and from the final fusion PCR, I can see a band on an agarose gel of the correct size (~1.2kb). For the fusion PCR I have used Roche Expand Long template PCR system (because it has good proof reading ability). I then gel extracted the above product and set up both 1hr and overnight ligation reactions with PGEMT-easy vector. I transformed the ligations into 15ul of XL10 Golds and with both ligation reactions I only get a few colonies for my positive control (PGEMT-easy positive control insert) and no colonies on my transformation plates (1:1 insert to ratio and 3:1). I am not sure why.
I have done similar PCR's and transformations before and have had success. Because the positive control and other transformations I have done at the same time have been successful, I am pretty confident the XL10 golds, antibiotics etc are fine.

Any ideas will be greatly appreciated.

Thanks!

I don't think Expand adds the 3' A necessary for TA cloning to work. Your PCR reaction is very far from "long" so the Expand is unnecessary. You can treat your PCR product with Taq + dATP to add the A's, or (in my opinion easier) use a Pfu + Taq mixture and redo the last PCR reaction.

Thanks! I will try A-tailing. If that fails I will try re-doing the last PCR