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 litre

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.