Yeast Plates
Hahn Lab - Methods - Yeast Plates
Yeast Plates
Hahn Lab
Last modified Tue, Oct 6, 1998
Amino Acid Mix (40X) Use 0.6 g/liter
Tyrosine 2 g
Arginine 4 g
Serine 2 g
Valine 2 g
Threonine 4 g
Isoleucine 2 g
Phenylalanine 2 g
Aspartic Acid 2 g
Proline 2 g
Glucose Complete Plates (1 liter):
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 liter):
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 liter):
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.
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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.
Yeast Plates
Hahn Lab
Last modified Tue, Oct 6, 1998
Amino Acid Mix (40X) Use 0.6 g/liter
Tyrosine 2 g
Arginine 4 g
Serine 2 g
Valine 2 g
Threonine 4 g
Isoleucine 2 g
Phenylalanine 2 g
Aspartic Acid 2 g
Proline 2 g
Glucose Complete Plates (1 liter):
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 liter):
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 liter):
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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