I have been trying knocking out so called non essential gene (pagP) from E.coli chromosome using datsenko and wanner method but it's not knocking out. On testing the so called knockout strains with PCR by using primers that lies out side of pagP both the bands of gene and selection marker appear on the electrophoresis. I am suspecting there might be any kind of duplication is going on with the gene during knock out process as a result one copy is getting replaced with selection marker where as other one is remaining intact.
I would be grateful if any one have any ideas regarding the protocol to determine whether any kind of duplication is going on or not during knocking out process? Can this be a case of meridiploidy?
Similarly, if a pagP gene is not wanting to knock out from the chromosome then it might be possible that it is essential to the E.coli Is there any way that I can test whether this gene is essential to E. coli or not?
Thank you in advance.
protocol to determine an essential gene and meridiploidy
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