Hi everyone !
'I've been working on a gene knockout in a BC cell line. 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 cell and KO cell ?
Thank you in advance for your reply.
Hey,
I would first do Sanger sequencing on the DNA from your clones, since you are doing a genomic KO (CRISPR?). Then you will know what happened exactly with your sequence of interest. Maybe the cells repaired in a matter that there is a small substitution and qRT-PCR runs normally, but the change occur within the epitope recognized by the antibody and you see no bands in Western Blot.
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