I need to prepare YPD plates containing hygromycin, but I don't know which concentration I can use. I'll select transformants of Hansenula polymorpha.
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1$\begingroup$ Presumably this will depend upon the promoter that is driving expression of the marker, and the copy number of the plasmid. Do you know anything about either of these parameters? $\endgroup$– Alan BoydCommented Apr 10, 2013 at 16:08
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$\begingroup$ First I'll linearize this plasmid. because I want it to be integrated into the genome. (linearized DNA integrates to the genome in H. polymorpha). So there will be no additional copies. And tef1 promoter is present before hygromycin gene. $\endgroup$– ArmacinoCommented Apr 10, 2013 at 20:14
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According to this site, for yeast (I assume this means Saccharomyces cerevisiae) you should use 50 µg/ml to 200 µg/ml; for fungi (!) use 100 µg/ml to 300 µg/ml. The site also stresses the importance of the pH of the medium.
I think that, unless a Hansenula expert comes along, you will have to try an initial experiment to measure the sensitivity of untransformed cells across the range indicated. Then you can try selecting resistant cells at a concentration somewhat higher than that which kills untransformed cells. Finally, you can check to see how resistant the transformants are, to enable you to decide on a useful concentration for routine selection of transformants.
answered Apr 10, 2013 at 21:40