I'm interested in using the Bochner-Maloy Tetracycline-sensitivity positive selection strategy, described many years ago (works with different enterobacteriaceae). In this method, culture medium must be supplemented with about 6 ml/L of a Fusaric acid stock solution (2 mg/ml). Do anyone know in which solvent this stock solution should be prepared? For how much time can the stock solution be stored? At which temperature?
I've tried this method a few times (Didn't really work with my bugs, but they aren't enterobacteriaceae). I dissolved the fusaric acid in water and stored at 4 deg C. I didn't do a lot of experiments with this so I don't know how long this is stabile at 4. You could always freeze at -20 in smaller volumes to minimize freeze thaw cycles. Fusaric acid is soluble in Ethanol, and at -20 it won't freeze, so you wouldn't have the freeze/thaw issues so much; but then you are adding Ethanol to the medium. There was another method for Tetracycline counter-selection that used Nickel Ions (Podlosky, et al 1996 PMID 8954882). I had better luck with this method for my bacteria. Hope that's helpful.
Thanks for your tips!, gotta try both the Nickel chloride and Fusaric acid strategies.
Hi, maybe you know about it but, you may check the paper by Li (Xin-tian), et al. published in Nucleic Acids Research Advance (2013) , "positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in E. coli".
They provide some useful information about plate and selection medium.
Best regards
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