Good morning everyone
I have created the pseudomonas oryzihabitans mutant via Gibson assembly (homologous recombination) with the suicidal vector pK18mobsacB . And now I am trying for complementation of the gene with the same vector i.e pKmobsacB but I am not able to transform the plasmid in the pseudomonas mutant. Please suggest what could be the possible reasons for transformation failure or there is a problem with the vector as I am using same vector for deletion and complementation? As I searched many papers but no one have gone for gene deletion and complementation with the same vector.
Are you certain that the DNA prep of your complementing plasmid is really of high quality, integrity and sufficient concentration? And are you sure your competent cells (are you using electroporation, chemical competence, conjugation) are working well?
Thank you for the gentle response; Yes I am very much sure about the competent cells and also of the plasmid (Conc. 400 ng/microl; 1.8 260/280 ratio), which I have crosschecked through the double digestion in which I got the 2 specific band of vector (pKMobsacB) and insert (targeted gene) for complementation.
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