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).

---