How to Make Agar

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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