DESCRIPTION 5-FOA Counter-selection with 5-FOA is a commonly used molecular genetic method for yeast. Curing yeast strains of plasmids, plasmid shuffle, allelic replacement, and two-hybrid screens are standard methods employing 5-FOA (Boeke, J.D. et al. 1984 and 1987; Vidal, M. 1997). The nontoxic 5-FOA compound is converted to toxic 5-flurouracil in yeast strains expressing a functional URA3 gene (orotine-5’-monophosphate decarboxylase), whose normal cellular function is in the synthesis of uracil. Practically, strains that are phenotypically Ura+ become Ura- and 5-FOAR (resistant), after selection. The question of solubility is often raised in relation to 5-FOA at Zymo Research from customers using our ultra pure 5-FOA powder (>98%). This nontoxic compound is only slightly soluble in water and reliable useful data on solubility is generally lacking from chemical companies. The ability of 5-FOA to dissolve in different commonly used solvents was tested to find a useful solvent that is compatible with yeast selection methods to provide a convenient liquid solution for making counter selection media. It was found that there was appreciable solubility of 5-FOA in dimethyl sulfoxide (DMSO). The solubility of 5-FOA in DMSO was found to be greater than 150 mg/ml, allowing concentrated solutions to be made. A convenient 100 mg/ml (100X) solution and a 2X synthetic complete (SC: yeast nitrogen base, ammonium sulfate, complete amino acids, glucose) liquid media containing 5-FOA are now available for use. All 5-FOA formulations are stable, convenient, and easy to use.

Yeast strains that are auxotrophic for uracil (ura3-1) were tested for their ability to grow on 5-FOA containing media. Three strains were tested: wt alone (YZ1), wt with a URA3 marked low copy plasmid (YZ2), and a mutant strain with a deletion of an essential gene (EG) that could not lose a complementing URA3 plasmid (YZ3). From left to right are synthetic complete glucose medium (SC): 1. SC, synthetic complete no 5FOA; 2. Standard-SC-5-FOA (SC-5-FOA made from ultra pure 5-FOA powder, 1 g/liter) 3. SC-5-FOA made from 100X 5-FOA solution; 4: SC-5-FOA made from 2X SC/5-FOA. Top: Yeast strain: YZ1 wild-type, Ura- (wt, ura-3-52), Right: Yeast strain: YZ2, wt carrying a low copy, URA3 plasmid alone, and Left: Yeast strain: YZ3:EG, containing the complementing plasmid (pRS316: EG, URA3, CEN). The counter selection against strain YZ3 was evident for all media containing 5-FOA with no 5-FOAR colonies evident (see left panels, YZ3: 2, 3, and 4). Cells from control strains YZ1 and YZ2 were able to grow on 5-FOA media.