Anybody could obtain transfectant cells of Leishmania major Friedlin with the cassette of G418?. I don’t have any problem with others cassette (like Hygromycin and Blasticidin) but with neomycin it´s impossible, the culture don´t growth. Any suggestion? I use 10 ug/ml with M199 medium.
Hi, I've never had problems with neomycin selection in L. major FVI. I routinely grow transgenic promastigotes in M199 containing 40 ug/ml G418 (Life Technologies). Are there other problems with your construct e.g. expressing a toxic gene? Other suggestions: check the sequence of your NeoR gene and the 3' UTRs or try a different brand of G418?
The G418 activity may be quite different depending on the manufacturing batch. Every time that I purchase a new lot I test the drug activity performing a kill-curve and calculating the LC50. To select and grow the transfectants I use 4 x DL50 for M199 liquid medium and 8 x DL50 for M199 semi-solid plates.
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