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 祄 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 | 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 祃 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
All stock solutions can be autoclaved for 15 minutes at 121癈, 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 | 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 |
Filter sterilize and store at room temperature.
SCE/ DTT/ Lyticase (for 40 ml, prepare fresh each time):
40 ml SCE
300 祃 2 M DTT
5600 units Lyticase
SCE/2-ME/Lyticase (2 ml)
2 ml SCE
16 祃 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 礸/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 祃 | 3 mM |
| 12M 2-Mercaptoethanol | 25 祃 | 3 mM |
| Nonidet P-40 | 100 祃 | 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 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.
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