This reagent is used for negative selection of ura- cells. It is an analog of a metabolite in the uracil metabolic pathway which, in the presence of URA3 creates a compound that is toxic to yeast. Our supply of 5-FOA is stored in the dessicator on the bottom shelf of the -20 C freezer in the ice room. The dessicator is very slippery and heavy! Bring a strong buddy to help you.
Manufacturer's web page for 5-FOA.
Making 5-FOA Plates (1L)[]
Materials:[]
- 500 mL beaker
- Thermometer (measures greater than 60 degrees C)
- 2 L Flask
- 1000 mL Graduated Cylinder
- Filter Sterilizer (Steri-Cup)
- Screw Cap bottle w/ orange top (250mL)
| Reagents | Amount |
|---|---|
| Agar | 20 g |
| Ammonium Sulfate | 5 g |
| Yeast Nitrogen Base (YNB) w/o AA and AS | 5 g |
| Nutritional (His, Leu, Lys) | 1.4 g (of each) |
| Ura Powder | 20 mg |
| Glucose (50%) (After Autoclaving) | 40 mL |
| 5-FOA | 1 g |
[]
Making the 5-FOA: In the 500 mL beaker add 1g of 5-FOA to 210mL of ddH2O. Heat to 60 degrees C and stir to completely dissolve. Filter sterilize this before adding to liquid agar. (If you do not know how to filter sterilize please ask!)[]
Making the agar: In the 2 L flask, mix in 750mL of ddH2O[]
- 20g Agar
- 5g YNB w/o AA and AS
- 5g Ammonium Sulfate
- 1.4g each of His, Leu, and Lys
- 20mg Ura
Following the instructions of the pH meter, bring the solution to pH = 5.8
Autoclave
Cool to 55 degrees C
Add 40mL Glucose (50%)
Add all of 5-FOA to liquid agar