I have tried to perform conjugation of E coli having GUS transposon(TP003) with bacillus , Burkholder strains, what I have observed that after mixing both the antibiotic selective strains..with E coli when we plate the mixture in selective plates we are getting lawn of colonies instead of single colonies?? I am trying to understand what is wrong in the procedure, I am following standard protocols, but not getting success, please help me to solve the issue, the bacillus and Burkholderia I have made rifampicin resistant and E coli has ampicillin and streptomycin selection.
Could do describe the E.coli donor you are using? and the conjugation parameters you are using? are you using dapA or hemA mutants of E.coli to allow post conjugation counterselection?
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