Gene Knockout efficiency ?
Hi everyone !
'I've been working on a gene knockout in a Bacterial. The KO efficiency was explored for the first time after the limiting dilution and cell cloning by Western blotting. Some clones had no target protein band. But when exploring by by qRT-PCR,for the same clones(with no bands for the target protein), I can still find the mRNA expression.
My questions:
1/ In this case, how can I judge if my KO was successful or not?
2/ Does this imply any issues when moving to the next step: RNA sequencing comparing control Bacterial and KO Bacterial?
The answer depends on how was your KO done. It is possible that your strategy have eliminated protein expression while the RNA is still there, for example if you deleted the translation initiation site or introduced a stop codon rather than deleting the entire gene. If this is the case than having the RNA is not an issue. If feasible you can try to design qPCR primers targeting the deleted region in order to verify there is no wt transcript left.
Why don't you first confirm that you have actually constructed the knockout allele either by PCR or sequencing before you worry about levels.
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