get of ura3 mutant of a yeast - (Jul/17/2008 )
I tried to get ura3 mutant of a yeast by the combination of EMS or MNNG mutagenesis and 5-FOA selection and could get canditates that can grown on 5-FOA plate. But, all the candidates could grow on medium lacking uracil. Several condition changes also gave the same results. So, I would like to get it by other method. Does anyone know other mehods that give ura3 mutants?
You could try knock out the ura3 gene using gene targeting. THis method is alot more trouble some than mutagenesis, but I am more in favour of this method as you can get a clean and complete removal of the ura3 gene. FOA selection would still be used as part selection, although it can use an antibiotics marker or PCR or southern blot as a screening method.
Just wondering, have you optimised the concentration of FOA for your yeast species? (ie a kill curve).