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.