5-FOA Can agar plate media
MATERIALS
Reagents
5-Fluoroorotic acid (5-FOA; US Biological)
Bacto agar (Becton, Dickinson and Company)
Canavanine sulfate (Can; Sigma)
Exclude arginine and uracil from the recipe. Alternatively, a complete synthetic media dropout powder lacking uracil and arginine is available commercially (US Biological D9537-03).
Glucose (40%, w/v)
Uracil (Sigma)
Yeast nitrogen base without amino acids (Becton, Dickinson and Company/Difco)
Yeast nitrogen base without amino acids comes with and without ammonium sulfate. This protocol is designed for the form containing ammonium sulfate.
Equipment
Autoclave
Filters, sterile, 0.22-μm (e.g., Corning)
Flasks, autoclavable
Petri dishes, plastic, 14.5-cm diameter
Stir bars
Stir plate, magnetic
Vacuum filtration system
METHOD
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1. Add 24 g of Bacto agar and 750 mL of water to an autoclavable flask containing a magnetic stir bar. Autoclave the mixture.
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2. To a separate flask containing a stir bar, add the following to make 250 mL of nonautoclavable mix:
Reagent Amount to add (for 1 L final volume of 5-FOA Can agar media) 5-FOA 1 g Canavanine sulfate 60 mg Dropout powder or mix 2.0 g Glucose (40%, w/v) 50 mL Uracil 50 mg Yeast nitrogen base without amino acids 6.7 g Water 200 mL Avoid boiling the nonautoclavable mix for long periods of time.
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3. Filter-sterilize the nonautoclavable mix.
If the nonautoclavable mix is ready before it can be added to the autoclaved agar, keep the nonautoclavable mix warm (e.g., in a 50°C incubator) to prevent rapid solidification of the autoclaved agar solution.
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4. Cool the autoclaved agar to 60°C or cooler. Combine with the sterilized nonautoclavable mix. Stir on a magnetic stir plate.
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5. While still warm, pour the combined media into 14.5-cm diameter plastic Petri dishes. Allow to solidify.
Media can be tested by checking the growth of strains with the genotypes URA3 CAN1, URA3 can1, ura3 CAN1, and ura3 can1.
