Hi!
I'm trying to create a deletion mutant using pEX18 Gm. I amplified my gene, cloned into pEX18 and with electroporation (1600v), my plasmid vector integrated into P.aeruginosa genome.
But now i'm having trouble with sacb counterselection. I've used %15 and %20 sucrose but still suicide vector is in P. aeruginosa genome. I tried to verify sucrose colonies on LB Agar and LB Gentamisin Agar (Gm 20 mg and Gm 60 mg) but still there isn't a mutant colony. How can I overcome this problem?
Thank you.
Is the problem that you have too many colonies (everything is growing) or that you have no colonies?
Colonies grow on No Salt LB %15 sucrose but after that I can't verify them on LB Gentamicin and LB agar. Theoretically colonies shouldn't grow on LB Gentamicin. But they are growing.
Thanks for help.
Two suggestions, first just screen a bunch of colonies from the sucrose plate (like 100 or so) to see if you can find it.
A second suggestion is to work with a few different plasmid integration clones. You might have gotten an unusual integrant that isn't behaving correctly, so just starting from a different integrant clone might work more normally. I've seen this happen on occasion.
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