Hi everyone,

I am trying to get a 3xflag tag (around 100bps) onto the end of a sequence of interest. I have the vector (3kbp), with the sequence already in it and an EcoRV site where I want to insert my tag. Both vector and insert are gel purified, and I treat the linearised vector with NEB antarctic phosphatase to stop re-ligation. I have tried to assemble the plasmid with Takara Infusion, NEB HiFi DNA assembly and a T4 ligase, increasing the molar ratio up to 1:10 but nothing has worked and the few colonies I have are just religated with some random bps in the gap.

I read that using UV light to cut out my gel band during gel purification might be affecting the quality of the insert. Does anyone have any experience of this or any other suggestions?

Thanks in advance.