Yeast Media, Solutions and Stocks

Updated Jan. 17, 1991

Jim Howe and C. Helms

Yeast Media:

Note: Synthetic complete medium can be prepared by adding media supplements (see below).

Medium using 6.7 g yeast nitrogen base without amino acids (Difco # 0919-15) can also be prepared using 1.7 g yeast nitrogen base
without amino acids and ammonium sulfate (Difco #0335-15) and 5.0 g ammonium sulfate.

AHC Medium (a selective medium for growing yeast strain AB1380, a host for YACs)

6.7 g yeast nitrogen base (without amino acids) Difco# 0919-15

10 g casein hydrolysate acid

20 mg adenine sulfate (or 10 ml adenine sulfate stock solution, see supplements below)

1 liter dH2O

pH to 5.8 with approximately 50 µm 12 N HCl

autoclave 30-45 minutes and cool to 65 degrees C, then add 50 ml sterile 40% glucose

AHC plates

Add 20 g Bacto agar to AHC medium before autoclaving

SD Medium (a synthetic, minimal medium)

 6.7 g yeast nitrogen base (without amino acids) Difco# 0919-15

1 liter dH2O

autoclave for 30-45 minutes and cool to 65 degrees C, then add 50 ml of sterile 40% glucose

SD plates

Add 20 g Bacto-agar before autoclaving

Sorbitol-Agar Medium (1 liter)

(a 1 M sorbitol synthetic medium with a lower adenine concentration used to plate out transformed yeast cells)

182 g sorbitol

6.7 g yeast nitrogen base (without amino acids) Difco# 0919-15

Uses eleven of the synthetic media supplements (see below): adenine, arginine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, threonine, tryptophan, and tyrosine.

Pipet the prescribed ml amount of stock solution in the synthetic media supplements list for 1 liter of media, except for adenine: use 5
ml adenine (instead of 10 ml).

Add 900 ml dH2O, dissolve the ingredients, and pH the solution to 5.8 with 1 N NaOH.

Adjust the volume to 1 liter with dH2O, add 20 g agar and autoclave 30-45 minutes.

After autoclaving, add 50 ml sterile 40% glucose. Mix well and cool to 50 degrees C before pouring the plates.

Note: Because the medium will be used with spheroplasts (which are extremely sensitive to lysis with detergents) pre-rinse all glassware
at least 3 times before using.

Sorbitol-Agar Medium Top Agar

 Prepare Sorbitol-Agar medium with 25 g agar instead of 20 g. Autoclave 40 minutes/1L volume to sterilize.

Note: Because the medium will be used with spheroplasts (which are extremely sensitive to lysis with detergents) pre-rinse all glassware
at least 3 times before using.

Sporulation Medium Plates

Diploid strains will undergo several divisions on this medium and then sporulate after 3 -5 days incubation. Sporulation of auxotrophs is
usually increased by adding the nutritional requirements (see synthetic media supplements, below) to the medium at 25% the normal
levels.
1% potassium acetate 10 g
0.1% yeast extract 1g
0.05% glucose 0.5 g
2% agar 20 g
distilled water 1000 ml
To induce diploid strains to sporulate after 18-24 hours without vegetative growth, use this medium without the yeast extract and glucose.

YPD Medium (an enriched, non-selective yeast growth medium)

1. 10 g yeast extract
2. 20 g Peptone
3. 1 liter dH2O
4. adjust pH to 5.8 with approx. 50 µl 12 N HCl
5. autoclave 30-45 minutes and cool to 65 degrees C, then add 50 ml of sterile 40% glucose

YPD plates

Add 20 g Bacto agar before autoclaving

Synthetic Media Supplements

 All stock solutions can be autoclaved for 15 minutes at 121°C, and stored for extensive periods. Some stocks should be stored at room
temperature (indicated below by a *) to prevent precipitation, while the others should be refrigerated. When using threonine and aspartic
acid, add after autoclaving the media. Use the HCl salts of amino acids wherever applicable.
Stock solution ml stock for
Constituent Final mg/L per 100 ml dH2O 1 liter media
adenine sulfate 20 200 mg* 10
uracil 20 200 mg* 10
L-tryptophan 20 1g 2
L-histidine-HCL 20 1g 2
L-arginine-HCL 40 1g 4
L-methionine 20 1g 2
L-tyrosine 50 200 mg 25
L-leucine 60 1g 6
L-isoleucine 60 1g 6
L-lycine-HCL 50 1g 5
L-phenylalanine 50 1 g* 5
L-aspartic 100 1 mg 10
L-glutamic acid 100 1 g* 10
L-valine 150 3g 5
L-threonine 200 4 g* 5
L-serine 400 8g 5
* Store at room temperature

Solutions and stocks:

40 mM EDTA, 90 mM 2-Mercaptoethanol (2-ME)

40 ml 0.5M EDTA, pH 8.0

3.8 ml 2-Mercaptoethanol (12M stock)
 456.2 ml sterile ddH2O
500 ml Store at room temperature.

5X HPB (High Phosphate Buffer) (for 1 liter)

1. 146.1 g NaCl
2. 134.0 g Na2HPO4
3. 9.3 g Na2EDTA
4. pH to 7.0 with concentrated HCl
5. Autoclave to sterilize

SCE (100 ml)

Final concentration
2M sorbitol 50 ml 1.0 M
1M sodium citrate 10 ml 0.1 M
0.25M EDTA, pH7.0 24 ml 60 mM
sterile ddH2O 16 ml
------
100 ml
Filter sterilize and store at room temperature.

SCE/ DTT/ Lyticase (for 40 ml, prepare fresh each time):

1. 40 ml SCE
2. 300 µl 2 M DTT
3. 5600 units Lyticase

SCE/2-ME/Lyticase (2 ml)

1. 2 ml SCE
2. 16 µl 2-ME
3. 0.2 mg Lyticase
Mbo I Buffer

1. 100 mM NaCl
2. 10 mM Tris-HCl, pH 7.4
3. 10 mM MgCl2
4. 1 mM Dithiothreitol
5. 100 µg/ml bovine serum albumin

Miniprep Lysis Buffer

Final concentration
1M Tris-HCl, pH 7.4 5 ml 50 mM
0.5M EDTA, pH8.0 5 ml 25 mM
5M NaCl 10 ml 500 mM
1M MgCl2 300 µl 3 mM
12M 2-Mercaptoethanol 25 µl 3 mM
Nonidet P-40 100 µl 0.1 %
10% SDS 10 ml 1.0 %
Bring volume to 100 ml with sterile ddH2O.

Filter sterilize and store at room temperature.

Large Scale Prep Lysis Buffer

0.5 M Tris-HCl, pH 9.0 2.5 ml 2 M Tris-HCl stock

3% sarkosyl 3.3 ml 10% sarkosyl stock
0.2 M EDTA 4 ml 0.5 M EDTA stock
Adjust volume to 10 ml with sterile ddH2O.

1 mM Tris/1% Sarkosyl (for 1 liter)

1. 1 ml 1M Tris-HCl, pH 7.5
 2. 100 ml 10% sarkosyl
3. 899 ml dH2O

200 mM Tris/2X SSC (1liter)

1. 200 ml 1M Tris-HCl, pH 7.5

2. 100 ml 20 X SSC
3. 700 ml dH20

Yeast prehyb/hyb solution (for 10 ml)

1. 7 ml sterile dH2O
2. 2 ml 5X HPB
3. 1 ml 10% sarkosyl

Yeast Lysis Buffer

1. 50 mM EDTA
2. 1% sarkosyl
3. 10 mM Tris-HCl, pH 8.0
4. 1 mg/ml proteinase K