Also see recipes for drugs and DNA damaging agents. These are our standard media; other pombe groups may have different preferences. Fission yeast can be grown on S. cerevisiae media (YPD and SD). However, the cells are not as happy on peptone (the P in YPD), and our experiments suggest that standard yeast nitrogen base (SD) contains enough thiamine to repress the commonly used nmt promoter. But for general culturing purposes, S. cerevisiae media will work fine.
Prepared media can be obtained as a powdered homogenate from several biotech firms. While these can be more expensive than making it yourself, they are time-saving, and this may be more practical especially if your use is occassional. You can also grind your own medium to make an equivalent powder. Note that all media should be sterilized by autoclaving (15 or 20min max to avoid caramelizing the sugar), although liquid media can be filter sterilized.
YES: Yeast Extract with supplements
amt | component | final conc |
---|---|---|
5 g/l | yeast extract | 0.5% w/v |
30 g/l | glucose | 3.0% w/v |
supplements: 225 mg/l adenine, histidine, leucine, uracil and lysine hydrochloride. | ||
Solid media is made by adding 2% Difco Bacto Agar |
Variations
Yeast extract + phloxin B (YEP – Checking ploidy) YES + 5 mg/l phloxin B (Sigma No. P 4030). Add when the medium has cooled below 60*C from a 5g/l stock solution in sterile distilled water. |
YSO As YES, but at 2g/l cas-amino acids to final concentration of 0.2%. (No adenine). |
EMM: Edinburgh minimal medium
amt | component | final conc |
---|---|---|
3 g/l | potassium hydrogen phthallate | 14.7mM |
2.2 g/l | Na2HPO4 | 15.5 mM |
5 g/l | NH4Cl | 93.5 mM |
20 g/l | glucose | 2% w/v |
20 ml/l | salts | |
1 ml/l | vitamins | |
0.1 ml/l | minerals | |
Solid media is made by adding 2% Difco Bacto Agar |
PMG: Pombe Glutamate medium
amt | component | final conc |
---|---|---|
3 g/l | potassium hydrogen phthallate | 14.7mM |
2.2 g/l | Na2HPO4 | 15.5 mM |
3.75 g/l | L-glutamic acid, monosodium salt (Sigma G-5889) | |
20 g/l | glucose | 2% w/v |
20 ml/l | salts | |
1 ml/l | vitamins | |
0.1 ml/l | minerals | |
Solid media is made by adding 2% Difco Bacto Agar PMG is recommended because of more even growth of Ura+ and Ura- strains, and is also compatible withG418 selection. Not to be confused with EMM(low)Glut, below, which uses a limiting amount of glutamate (nitrogen) to induce sporulation. |
Variations
For intermediate repression (described in this reference): 0.05uM thiamine (0.016ug/ml).
Why buffer with phthallate? Paul Nurse says this choice was made to reduce clumping, which was a particular problem with cdc mutants.
Stock Solutions
filter sterilize and store at 4*C
50x Salt stock
amt | component | final conc |
---|---|---|
52.5 g/l | MgCl2.6H20 | 0.26 M |
0.735 g/l | CaCl2.2H20 | 4.99 mM |
50 g/l | KCl | 0.67 M |
2 g/l | Na2SO4 | 14.l mM |
1000x Vitamin stock
amt | component | final conc |
---|---|---|
1 g/l | pantothenic acid | 4.20 mM |
10 g/l | nicotinic acid | 8l.2 mM |
10 g/l | inositol | 55.5 mM |
10 mg/l | biotin | 40.8 uM |
10,000x Mineral stock
amt | component | final conc |
---|---|---|
5 g/l | boric acid | 80.9 mM |
4 g/l | MnSO4 | 23.7 mM |
4 g/l | ZnSO4.7H2O | 13.9 mM |
2 g/l | FeCl2.6H2O | 7.40 mM |
0.4 g/l | molybdic acid | 2.47 mM |
1 g/l | KI | 6.02 mM |
0.4 g/l | CuSO4.5H2O | 1.60 mM |
10 g/l | citric acid | 47.6 mM |
MB media
This is a very stringent minimal medium that is used for lithium acetate transformation protocol
amt | Compound |
---|---|
0.5 g/l | KH2PO4 |
0.36 g/l | K-acetate |
0.5 g/l | MgSO4.7H2O |
0.1 g/l | NaCl |
0.1 g/l | CaCl2.2H2O |
5 g/l | (NH4)2SO4 |
500 ug/l | H3BO4 |
40 ug/l | CuSO4.5H2O |
100 ug/l | KI |
200 ug/l | FeCl3.6H2O |
400 ug/l | MnSO4.H2O |
200 ug/l | Na2MoO4.2H2O |
400 ug/l | ZnSO4.7H2O |
5g/l | glucose |
10ug/l | biotin |
1mg/l | calcium pantothenate |
10mg/l | nicotinic acid |
10mg/l | myo-inositol |
150 mg/l | uracil for ura4- strains or |
150 mg/l | leucine for leu1- strains. |
Filter sterilize |
Mating/Sporulation media
Autoclave as usual
ME: Malt extract
amt | component | final conc |
---|---|---|
30 g/l | Bacto-malt extract | 3% (w/v) |
Supplements added as for YES except lysine. Adjust to pH 5.5 with NaOH. | ||
Solid media is made by adding 2% Difco Bacto Agar |
SPAS mating media
amt | component | final conc |
---|---|---|
10 g/l | glucose | 1% (w/v) |
1 g/l | KH2PO4 | 7.3mM |
1 ml | 1000x vitimins | (w/v) |
supplements: 45 mg/l adenine, histidine, leucine, uracil and lysine hydrochloride (1/5 normal). | ||
Solid media is made by adding 3% Difco Bacto Agar |
Growth on nonfermantable carbon sources
Recently (20 Nov 07) a discussion on the pombelist mailing list addressed growth on nonfermentable carbon sources. S. pombe grows very poorly on non-fermentable carbon sources. People report various results, which I summarize here:
Strains will grow better if ura4+
You need to include replacements for citric acid cycle intermediates. Kurt Runge quotes, “fats burn in the flame of carbohydrates”, but done without glycosis. and recommends the old Gutz method of 0.37% mono sodium glutamate
Suggested recipes
1% yeast extract, 0.5% casaminoacids, 3% glycerol, 3% ethanol, 40 microg/ml adenine. (Nathalie Bonnefoy)
use galactose as non-fermentable carbon source, in 1% yeast extract, 1% bactopeptone, 0.1% glucose, 2% galactose, 20 microg/ml adenine (the peptone here comes from an old cerevisiae recipe, it could be eliminated). But you can also try galactose without the 0.1% glucose (they will be slower to start growing), or you can reduce the glucose to 0.05%.(Nathalie Bonnefoy). Charlie Hoffman notes that sucrose, maltose, or raffinose may work.
0.67% Yeast nitrogenbase with 150 mg/l supplements for auxotrophies plus 3% glycerol + 0.37% mono sodium glutamate (from a 7.4% MSG filter-sterilized stock). After 2 weeks, cells reach about 4e7 cells/ml by hemocytometer counts, where as 3% glucose gives 6 e7 in similar medium without MSG. (Kurt Runge)
Yeast Extract -dextrose + 3% glycerol as a growth assay for S. pombe. Wild type colonies in 3-4 days. (Nate Blewitt,)
liquid 0.5%YE + glycerol (2%? 3%?) without any glucose. (Peter Fantes)
EMM doesn’t work.
Thanks to Peter Fantes, Charlie Hoffman, Nate Blewitt, Nathalie Bonnefoy , and Kurt Runge for responses quoted here.