PCR to confirm double crossover - (May/16/2011 )

Hi,

I'm trying to generate a knockout in M.smegmatis. I created the merediploid strain, and then used a suicide plasmid with sacB to generate my double crossover. I had a few double crossover candidates that were resistant to sucrose and I screened them using PCR with primers that would bind outside of the homologous regions I used to generate my DCO. When I looked at the gel, I had a single band that was the appropriate size for my WT DNA, but all of my DCO candidates had both a band that would be expected for the WT and then a band that I expected to find if the double crossover was successful. What is going on? My primers cannot bind to the plasmid I used for the merediploid. Does this mean that somehow the bacteria are harboring the suicide plasmid without the double crossover event? Is there any other possible explanation?

Thanks so much!!

Hi,

If I am understanding you correctly, your gel shows a WT band and a smaller knock-out band (DCO). To me, it would seem that your plasmid is still integrated and the isolates are still merodiploid. How did you know they were merodiploids in the first place? Also, how did you do your sacB selection? Did you plate? Grow culture, then plate? What sucrose% did you use?