First, PCR is commonly used. If it is positive, then sequence to further confirm.

PCR is very useful for confirming that a piece of DNA is physically present or absent. However, it may be the case that your knockout alters bacterial behavior in some way (this is usually why people do knock out experiments), so did your experiment present the functional change you expected? Is there a difference on Western? Can the knocked-out behavior be re-extablished by expressing the gene targeted on a plasmid?

Good Luck

G

Thank you very much Dear Gallagher. In fact I confirmed my gene knock out by sequencing. Surely there is a change in the phenotype (retarded growth). As for as, gain of function is concerned, I am working on it. However, I wanted to ask that only DNA sequencing is enough to confirm the results or I have to do western blotting or some other kind of techniques related to protein?

Hi again

Usually, the more evidence one has, the better. Western blotting though may not be possible if there isn't a good antibody (and you may have to establish this experimentally, despite what the catalogues may tell you ;) ). The thing that Western doesn't tell you is whether your target protein is working by itself or impacting others too - to get this information, I'm afraid it's a 2D protein gel. I'd not do this in my lab unless I was convinced that I had a *new* mechanism to investigate.

For now, the complementation experiment is the key one.

Good luck!

G