I have retrieved a plasmid (pucSIVGAG) that contains the insert Sivgag. I digested the plasmid using Sma I and did a gel purification to isolate the insert sivgag.

I am inserting sivgag into plasmids known as pcDNA 4/TO and pcDNA 3.1zeo+. I recently added Not I linkers to the ends of sivgag and purified with Promega PCR purification kit. I isolated the pcDNA vectors from gel purification and digested both insert and plasmid with Not I enzyme. I ligated the vector and inserts and transformed the ligation into EColi competent cells. I plated the cells on Carbenicillin LB Agar plates.

I have done this several times yet on all occasions there are no colonies that grow on the plates. If there happens to be colonies I run minipreps on them and I see that the vector has self-ligated and the insert is not being taken up. Is there something else I can do to get the insert to be taken up? Or is there something I am doing wrong?

Note: I have also already done treatment of shrimp alkaline phosphatase on the vectors to remove the phosphates and lessen the occurrence of self-ligation