a problem happened in my allele replacement experiment - (Jun/28/2012 )

I am doing an allele replacement of a gene in Salmonella. But every time the primary and secondary combination events occur on the same side, the right side of the mutation, so that it leads to the formation of wild-type recombinants instead of mutant recombinants. As you know, only when the primary and secondary recombination occur on the different side of the mutation site, it will leads to the mutant-type combinants. It seems that the sequence of the left side of the mutation have lost its function of allele replacement. But I've done the experiment twice using different mutant plasmids, but all of them have this problem. So I think there is another reason causing the problem.


Have you meet the similar problem? And how did you fix it? Thank you very much for your help!

Does anyone here have met the same or similar problem like me？