FOA培养基配置方法.docVIP

5-FOA (5-Fluoroorotic Acid) Medium and Agar Plate RecipeFollowing recipe makes 1 liter 5-FOA plates:Step-1: Autoclave 20 grams of agar in 750 ml double distilled water.Step-2: Add followings into a glass beaker, and bring to 250 ml with double distilled water. Ammonium Sulfate? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?5.0 g??Yeast nitrogen base without amino acid??1.7 g Dextrose? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ? 20 g??Necessary nutritional supplements (see note 3)??X g 5-FOA? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ???1.0 g??Warm up 5-FOA mixture in Step-2 at 55-65°C on a heat plate, stir until it dissolves ~30 min-1 h. The solubility of 5-FOA is very low in water; therefore, it is necessary to warm up the solution. Some manufacturers, i.e., US Biological, claim that 5-FOA is resistant to autoclaving. I did not try to autoclave it myself, but you can try it. After 5-FOA containing mixture in Step-2 is completely dissolved, sterilize it by filter. Place both melted agar and 5-FOA containing solution in a water bath (50-60°C) for one hour. Finally, add 5-FOA solution onto melted agar and pour your plates. Plates may be dried at RT for 48 hours and may be kept at 4°C for long term storage. Notes: 1.Do not forget to add Uracil into the plates at 20 mg/L of final concentration (Do not add too much Uracil!). 2.Never adjust the pH of 5-FOA containing medium. Only protonated form of 5-FOA is permeable. pH of your plates will be very acidic after adding 5-FOA. 3.Do not add more nutritional supplements than it is necessary; this reduces efficacy of 5-FOA plates. For example, you only need to add His, Leu, Lys and Ura supplements for strain BY4742. These supplements may be added directly from 100x stock solution. 4.5-FOA is very expensive reagent. I found that US Biological has the cheapest price for 5-FOA. 5.5-FOA is highly mutagenic to Candida albicans. This may also be the case for budding yeast. I suggest you do not leave yeast colonies on 5-F