How to Make Agar

Preparing Yeast Plates (The Hahn Lab, The Fred Hutchinson Cancer Research Centre and Howard Hughes Medical Institute, Seattle)

AMINO ACID MIX (40X) USE 0.6 g/LITRE

  • Tyrosine 2 g
  • Arginine 4 g
  • Phenylalanine 2 g
  • Threonine 4 g
  • Isoleucine 2 g
  • Aspartic Acid 2 g
  • Serine 2 g
  • Valine 2 g
  • Proline 2 g

GLUCOSE COMPLETE PLATES (1 LITRE):

  • 1.7 g Yeast Nitrogen base -amino acids and ammonium sulfate
  • 5.0 g Ammonium
  • Sulfate 1 liter H2O
  • pH to 7.0 using NaOH

Add:

  • 0.6 g Amino Acid mix (mix well before adding)
  • 20g Bacto-agar (Difco)

Autoclave 25 min.

Add:

  • 10ml (10/mg/ml) of any other required amino acids
  • 10ml 0.4% Adenine sulfate in 0.1M HCl if required
  • 10 ml 0.2% Uracil if required
  • 50 ml 40% glucose

YPD PLATES (1 LITRE)

  • Yeast Extract 10 g
  • Bacto peptone 20g
  • Bacto-Agar 20g
  • H2O 1 liter

Autoclave 25 min
Add:

  • 50 ml 40% glucose
  • 5 ml 0.4% Adenine sulfate in 0.1 M HCl if required
  • Add 10 ml 0.2% Uracil (only if plates will be used for tetrad dissection

5-FOA PLATES (0.5 LITRE)

Agar:

  • 15 g Bacto-agar
  • 300 ml H20 in 500 ml flask

Autoclave 25 min.

FOA mix:

  • 0.85 g Yeast Nitrogen base – amm. sulfate and amino acids
  • 2.5 g Ammonium sulfate
  • 0.3 g amino acid mix (from above)
  • 25 ml 40% glucose
  • 17.5 mg Uracil
  • 0.5 g 5-FOA
  • Any other required amino acids
  • 225 ml H2O

Microwave the above mixture for about 3 min until about 65 degrees. Stir on stir plate until dissolved (~10-15 min). Filter through 0.2 micron bottle top filter into sterile bottle. Add to autoclaved agar. Mix well and pour plates. Do not pH the plates as this will inactivate the 5-FOA.

ANTIBIOTIC YEAST PLATES:

G418 Plates
Use G418 for yeast containing the KanMX allele. Add to plates at 500 micrograms/ml for selection of gene disruptions using yeast transformation. Can use at 200 micrograms/ml for checking cells already containing resistance to G418.

Hygromycin B Plates
Use Hygromycin B at 200 microgram/ml. Add 4 ml 50 mg/ml (Roche) per liter

Nourseothricin (NAT) Plates
Use clonNAT (Werner BioAgents, Germany) at 100 micrograms/ml using a 1000x stock kept at -80 degrees.

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