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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.
Flouricacid
Manufacturer's web page for 5-FOA.

Making 5-FOA Plates (1L) Edit

Materials: Edit

  • 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

Edit

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!) Edit

Making the agar: In the 2 L flask, mix in 750mL of ddH2O Edit

  • 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

Pour plates