You using a single strand annealing protein like the Lambda Red beta/ICP8 system? Using a rolling circle replicon system to amplify your template as circular single stranded DNA is also an option.

Beatriz Rojo-Bezares it would be best if you provided more details about the organism, plasmid, insert, and homology for your system. Otherwise it is not possible to diagnose anything.

Also does low mean zero, or merely low? It is not clear what frequency you should expect to see, but often recombination events are low frequency.

Michael J. Benedik I will give you more details: I am trying to KO a gene in P. aeruginosa. I have constructed a vector containing the sequences up and downstream this gene, and with the selective marker tetC gene. After conjugation/electroporation, I find resisntant colonies, and in fact, some of them harbour the tetC gene. however, I cannot find any merodiploid, none of the resistant colonies integrate the vector in their genome.

I have tried with extra passages in selective media.

A couple of questions, is Tet marker between the upstream and downstream homologous So that is replaces the gene? And what is your basis for saying the vector is not present?

i presume the vector is non-replicating in PA?

do you get same results with both conjugation and electroporation?

Thanks for your answer Michael J. Benedik .

TetC is not between the upstream and downstream (that we have inserted in the MCS region of the vector, flanked by M13).

Effectively, the vector is not replicating in PA.

And yes, we have more or less the same results with electroporation and conjugation. In both cases I obtained colonies that seems to harbour the plasmid, however , the homologous recombination seem to not have take place. As I explained, I detect tetC in these colonies, but not the plasmid integrated.

It could be that you are just detecting plasmid template that went along with the cells after electroporation, or maybe entered the cell but did not recombine.

However it is still not clear by what criteria you are using to know the plasmid is present but not integrated?

Michael J. Benedik we use a PCR to test if the vector has been integrated in the genome (one primer in M13 and the other in the genome), and I do not obtain positive result for this PCR. However, I test also tetC PCR, and I obtain a clear, positive result.

From the way you describe your plasmid, there are two possible integration events, one in the upstream region and one in the downstream region. So a given pair of PCR primers are only going to give you a signal with one of those, the other would be too large. While in theory you should get equal numbers of both types, there might be some bias so that you are getting only one type. You might choose other primers to confirm.

OK, I try it.Thank you very much.