Colony PCR screen positive - insert digestion negative - (Dec/22/2009 )
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..
You have possibly been unlucky and introduced a methylation site for EcoKI methylase, which can inhibit SpeI digestion. This would explain your large fragment (which would be digested by XhoI but not by SpeI). The sequence to look out for would be A A C T A G T N N G T G C (in either the forward or reverse orientation).
phage434 on Dec 22 2009, 06:14 PM said:
Hi Phage, thanks fo the reply.
According to my primers design (which contain the SpeI site) there should be an ATTA cap following SpeI and a stop codon before. So I find it hard for this to be the reason.. Plus it wouldn't explain the 700bp band..
I guess a thing to do anyway would be single cuts or using another enzyme to release the insert (using the PGEM restriction sites).