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Yeast Media, Solutions and Stocks

Updated Jan. 17, 1991

Jim Howe and C. Helms

Yeast Media:

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)

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)

1 liter dH2O

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

SD plates

Sorbitol-Agar Medium (1 liter)

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

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	1 g
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)

10 g yeast extract
20 g Peptone
1 liter dH2O
adjust pH to 5.8 with approx. 50 µl 12 N HCl
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

		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	1 g	2
L-histidine-HCL	20	1 g	2
L-arginine-HCL	40	1 g	4
L-methionine	20	1 g	2
L-tyrosine	50	200 mg	25
L-leucine	60	1 g	6
L-isoleucine	60	1 g	6
L-lycine-HCL	50	1 g	5
L-phenylalanine	50	1 g*	5
L-aspartic	100	1 mg	10
L-glutamic acid	100	1 g*	10
L-valine	150	3 g	5
L-threonine	200	4 g*	5
L-serine	400	8 g	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)

146.1 g NaCl
134.0 g Na2HPO4
9.3 g Na2EDTA
pH to 7.0 with concentrated HCl
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

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

40 ml SCE
300 µl 2 M DTT
5600 units Lyticase

SCE/2-ME/Lyticase (2 ml)

2 ml SCE
16 µl 2-ME
0.2 mg Lyticase

Mbo I Buffer

100 mM NaCl
10 mM Tris-HCl, pH 7.4
10 mM MgCl2
1 mM Dithiothreitol
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 %

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

200 mM Tris/2X SSC (1liter)

200 ml 1M Tris-HCl, pH 7.5
100 ml 20 X SSC
700 ml dH20

Yeast prehyb/hyb solution (for 10 ml)

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

Yeast Lysis Buffer

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

References:

Sherman, F., Fink, G. R., and J. B. Hicks. (1986). Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Brownstein, B. H., Silverman, G.A., Little, R.D., Burke, D. T., Korsmeyer, S. J., Schlessinger, D., and M. V. Olson, (1989) "Isolation of single-copy human genes from a library of Yeast Artificial Chromosome clones". Science 244: 1348-1351.