bwschn01

I'm trying to create an isogenic mutant in bacteria using a suicide vector that should experience two recomination events. The first inserts an antibiotic resistant marker and the second should remove the gene I'm trying to delete as well as the antibiotic resistance marker. I get the first recombination event, but when I induce the second, I'm failing to see a mutant genotype. I can't figure out why. Any ideas on what could be causing this?

I've been tasked to come up with alternatives to generating an isogenic mutant bacteria for studying gene function and phenotype. I've come up with the conditional mutants. I'm hoping to find at least one more alternative. Can anyone direct me to a protocol or a paper? Thanks, bwschn01