Dear All,
Several times I've tried to transform a plasmid to Neisseria Subflava but I have never been successful .Anyone have a transformation protocol for transforming a plasmid to Neisseria Subflava?
If my experience with N. meningitidis is applicable to your case, check first (negative staining/TEM) if your strain is piliated; otherwise transformation will fail.
I just wondering if you have already tied other methods or not. In the attached paper I used common methods with differnt bactria and it works fine but I never tried Neisseria Subflava before
Article The pJan25 vector series: An enhancement of the Gateway-comp...
Another thing (and again, this may or may not be applicable to your situation) is that pathogenic Neisseria are apparently very sensitive to mechanical stress; I was never able to get transformants whenever I spread the transformation mixture on selection plates the way I'd do it with E. coli -i.e. with a glass spreader. Instead, I had to resort to using glass beads, or simply tilting the plates to spread the inocula evenly.
And yet another issue -is N. subflava a diplococcus? If it is, remember that each colony is not necessarily a single, clonal population. You have to pick transformants, dilute and replate to isolate pure colonies. Streaking is possible but tricky, due to that mechanical stress thing I mentioned above.
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