Need Help! Counter selectable marker sacB, rpsL - Need Help! Counter selectable marker sacB, rpsL (Apr/10/2008 )

I'm doing gene deletion in Vibrio harveyi strain. The problem is the wild type strain can't grow on 5%sucrose media(no salt) which will be use as a sacB second cross over selection.

I don't know if it because of LB+5% sucrose without salt is the problem. The strain can't grow without salt presence in the media.

So, I change to use rpsL marker (streptomycin-sensitive) instead. The problem still occured because we found rpsL sequence in this strain, so homologous often occured at rpsL site not an insertional site on plasmid. All the mutant we have has the mutation in rpsL site not interested gene.

Can we use sucrose plate with salt for sacB selection? We don't have any counter selectable marker other than sacB and rpsL......


Thanks

Look at the pKSS plasmid (Kast 1994) which allows counter selection on plates containing p-chloro phenylalanine. If you try it, make sure the pH of your medium is > 7.5 when doing the counter selection.

Why do we need to use no salt media for sacB selection? Can we add salt in it?