hope you can help with this. I amplified my gene of interest (2.5kb) with primers containing unique restriction sites (XhoI and SpeI). After amplifying by PCR with high-fidelity polymerase, I added A-tail and ligated into PGEM.
I picked colonies and did PCR screening using the same primers. In some colonies I got nice bands at 2.5kb, of the same intensity as a positive control colony.
But when I isolated DNA from those colonies and digested with XhoI/SpeI, I didn't get the 2.5kb insert! I only got a band at ~700bp and the vector backbone (which by the way seems a bit high, around 8kb instead of 3kb as it should be - nicked?).
I really have no idea what is going on. I've heard colony PCR screen can give you false positives (because of left over insert on the plate?), but the bands looked identical to control.
I digested 4ug with 20units of each enzyme for 1,5hr. Even if it didnt all get digested, I would expect something there (half amount of another plasmid gave me a nice band with the same conditions on the same run).
Any suggestions? I can always attach photos if that helps..
Edited by biobio, 22 December 2009 - 02:50 PM.