Which techniques should be used to confirm that gene has been knocked out and why? What about DNA sequencing of gene of interest by flanking primers?
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?
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