I create a mutant deleting a GOI and I complemented it using a vector carrying the removed GOI. I checked my strains by PCR using two pairs of primer, a pair for the removed gene and a second pair with which I should be able to amplify flanking regions of the gene.

I obtained two bands in the WT (GOI, flanking region+GOI), one band in the mutant (Flanking regions noGOI) and two bands in the complemented mutant (GOI, flanking regions noGOI).

Then I saved mutant and comp. mutant in glycerol stock -80 and I used it in my assay.

The function I'm studying was eliminated in the mutant but not restored in the complemented strain so I checked again the glycerol stock of the complemented strain by PCR and I found that there were no GOI anymore

I repeated the complementation from the beginning, triparental mating, antibiotic selection and PCR, I saved the candidate strain and I used it in my assay but again, the function due to the GOI was not restored and the PCR showed no GOI band.

I would suppose my mutant lose the vector very quickly but I always use it with specific antibiotic in order to maintain the vector inside. There is a 4 hours step in my assay in which I need to growth it in media with no antibiotic, could be that the problem?

Do you have any suggestion?