Dear Friends,
I am trying to make SifA deleted Salmonella Typhimurium strain using lambda red recombination procedure. I was following standard procedure using pKD46, pKD3 and pCP20 plasmids. After optimizing so many electroporation conditions, I have managed to get the expected SifA mutation only in wild type Salmonella Typhimurium strain. Attempts using Salmonella strains which already contain several other mutation were never be a success to me.
Does anyone have tried SifA mutation in Salmonella strains and recommend me some important steps that I can try.
I am particularly trying to optimize (1) pKD46 induction duration and arabinose concentration to be used and (2) electroporation conditions. So far I was trying following electroporation conditions; 2300 v, 25 uf, 200 ohms and 1 mm gap BTX cuvettes.
Kindly give some help to get this successful.
Thank you,
Kindly,
Amal.
If you have already constructed the marked gene with an antibiotic resistance marker in the wild type strain, why don't you just transduce the allele to the other strains selecting for the resistance gene.
Dear Michael J.Benedik,
Thank you very much for your valuable input. Professor,Can you provide me some more information about the allele transduction procedure. Does it involve bacteriophages?. If you can guide me to a protocol, it could be a big help at this moment.
Thank you,
Kindly,
Amal.
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