Hello, I am currently learning to generate Agrobacterium deletional mutants by homologous recombination. I already prepared the construct containing the up-and-down- fragments of the gene from Agrobacterium in a suicide plasmid (SacB, GmR) in E. coli S17. To obtain the deletional mutant I did bacterial conjugation between Agrobacterium and S17. The result of the first crossover seems fine since the Agrobacterium colonies I obtained contain the up and down fragments after I did the PCR check. However, in the second crossover, I thought at least I would see some colonies going back to wild type, but no bands are shown in all colonies I picked following the PCR. This experiment was actually already done by someone else in my lab, and she obtained the mutant. I used her mutant and the wild type of Agrobacterium as control. The PCR result of the control seems fine. So what is the possible reason for this failure, and is there any suggestion?
It is hard to diagnose what is going wrong without more details. But I would suggest you try again and use a few independent "first recombinants" and see how each behave. Sometimes you just get a weird colony for non-obvious reasons. But if you have someone who has done it already, why not ask them to help you trouble shoot, there might be something subtle that is wrong in the design.
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