{"id":308,"date":"2023-07-11T13:39:02","date_gmt":"2023-07-11T20:39:02","guid":{"rendered":"https:\/\/live-usc-dornsife.pantheonsite.io\/pombenet\/?page_id=308"},"modified":"2023-08-03T13:21:22","modified_gmt":"2023-08-03T20:21:22","slug":"media","status":"publish","type":"page","link":"https:\/\/dornsife.usc.edu\/pombenet\/media\/","title":{"rendered":"Fission Yeast Media"},"content":{"rendered":"\n\n \n \n\n\n\n\n\n\n\n
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Also see\u00a0recipes for drugs and DNA damaging agents.<\/a>\u00a0These 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.<\/p>\n

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.<\/p><\/div>\n<\/p><\/div>\n

 <\/p>\n

YES: Yeast Extract with supplements<\/h3>\n\n\n\n\n\n\n\n
amt<\/th>\ncomponent<\/th>\nfinal conc<\/th>\n<\/tr>\n
5 g\/l<\/td>\nyeast extract<\/td>\n0.5% w\/v<\/td>\n<\/tr>\n
30 g\/l<\/td>\nglucose<\/td>\n3.0% w\/v<\/td>\n<\/tr>\n
supplements: 225 mg\/l adenine, histidine, leucine, uracil and lysine hydrochloride.<\/td>\n<\/tr>\n
Solid media is made by adding 2% Difco Bacto Agar<\/strong><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/strong><\/p>\n

Variations<\/strong><\/p>\n\n\n\n\n
Yeast extract + phloxin B (YEP – Checking ploidy) <\/strong>
\n 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.<\/td>\n<\/tr>\n
YSO<\/strong>
\n As YES, but at 2g\/l cas-amino acids to final concentration of 0.2%. (No adenine).<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

EMM: Edinburgh minimal medium<\/h3>\n\n\n\n\n\n\n\n\n\n\n\n
amt<\/th>\ncomponent<\/th>\nfinal conc<\/th>\n<\/tr>\n
3 g\/l<\/td>\npotassium hydrogen phthallate<\/td>\n14.7mM<\/td>\n<\/tr>\n
2.2 g\/l<\/td>\nNa2HPO4<\/td>\n15.5 mM<\/td>\n<\/tr>\n
5 g\/l<\/td>\nNH4Cl<\/td>\n93.5 mM<\/td>\n<\/tr>\n
20 g\/l<\/td>\nglucose<\/td>\n2% w\/v<\/td>\n<\/tr>\n
20 ml\/l<\/td>\nsalts<\/a><\/td>\n<\/td>\n<\/tr>\n
1 ml\/l<\/td>\nvitamins<\/a><\/td>\n<\/td>\n<\/tr>\n
0.1 ml\/l<\/td>\nminerals<\/a><\/td>\n<\/td>\n<\/tr>\n
Solid media is made by adding 2% Difco Bacto Agar<\/strong><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

PMG: Pombe Glutamate medium<\/h3>\n\n\n\n\n\n\n\n\n\n\n\n
amt<\/th>\ncomponent<\/th>\nfinal conc<\/th>\n<\/tr>\n
3 g\/l<\/td>\npotassium hydrogen phthallate<\/td>\n14.7mM<\/td>\n<\/tr>\n
2.2 g\/l<\/td>\nNa2HPO4<\/td>\n15.5 mM<\/td>\n<\/tr>\n
3.75 g\/l<\/td>\nL-glutamic acid, monosodium salt (Sigma G-5889)<\/td>\n<\/td>\n<\/tr>\n
20 g\/l<\/td>\nglucose<\/td>\n2% w\/v<\/td>\n<\/tr>\n
20 ml\/l<\/td>\nsalts<\/a><\/td>\n<\/td>\n<\/tr>\n
1 ml\/l<\/td>\nvitamins<\/a><\/td>\n<\/td>\n<\/tr>\n
0.1 ml\/l<\/td>\nminerals<\/a><\/td>\n<\/td>\n<\/tr>\n
Solid media is made by adding 2% Difco Bacto Agar<\/strong>
\n 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.<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Variations<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n
Minimal supplemented with amino acids etc
\n EMM + 225 mg\/l supplements (ade, leu, his, lys, ura…) as required. We make stock solutions at 7.5mg\/ml (3.75 for uracil) and autoclave.<\/td>\n<\/tr>\n
Minimal low adenine (used to see red color associated with\u00a0ade6<\/i>\u00a0mutations)
\n Reduce adenine to 10 mg \/ l. (Concentrations from 7.5mg\/l to 30 mg\/l can be used) .<\/td>\n<\/tr>\n
Minimal low glucose (Used for Yeast transformations)
\n As EMM but 0.5% (w\/v) glucose instead of 2% (w\/v) .<\/td>\n<\/tr>\n
Minimal sorbitol (EMMS)
\n As EMM. Add 1.2M sorbitol.<\/td>\n<\/tr>\n
<\/a>Minimal (low) glutamate (EMMGlut, Used for sporulating diploids in liquid)
\n As EMM. Replace NH4Cl with 1 g\/l sodium glutamate (5.91 mM). Use 40mg\/l supplements. Note PMG has higher amount of glutamate.<\/td>\n<\/tr>\n
Minimal free phosphate (EMMP – Used for 32P-Phosphate labelling)
\n As EMM. Remove Na2HPO4 and replace potassium hydrogen phthallate with 2g\/l Na-acetate.3H2O (14.6 mM). The medium is adjusted to pH 5.5.<\/td>\n<\/tr>\n
Minimal + thiamine (to repress nmt promoter)
\n add 15uM thiamine (5ug\/ml; from filter sterilized 2000x stock at 10mg\/ml)<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

For intermediate repression (described in this reference<\/a>): 0.05uM thiamine (0.016ug\/ml).
\n Why buffer with phthallate? Paul Nurse says this choice was made to reduce clumping, which was a particular problem with cdc mutants.<\/p>\n

 <\/p>\n

Stock Solutions<\/h3>\n

filter sterilize and store at 4*C<\/p>\n

50x Salt stock<\/span><\/h4>\n\n\n\n\n\n\n\n
amt<\/th>\ncomponent<\/th>\nfinal conc<\/th>\n<\/tr>\n
52.5 g\/l<\/td>\nMgCl2.6H20<\/td>\n0.26 M<\/td>\n<\/tr>\n
0.735 g\/l<\/td>\nCaCl2.2H20<\/td>\n4.99 mM<\/td>\n<\/tr>\n
50 g\/l<\/td>\nKCl<\/td>\n0.67 M<\/td>\n<\/tr>\n
2 g\/l<\/td>\nNa2SO4<\/td>\n14.l mM<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

1000x Vitamin stock<\/span><\/h4>\n\n\n\n\n\n\n\n
amt<\/th>\ncomponent<\/th>\nfinal conc<\/th>\n<\/tr>\n
1 g\/l<\/td>\npantothenic acid<\/td>\n4.20 mM<\/td>\n<\/tr>\n
10 g\/l<\/td>\nnicotinic acid<\/td>\n8l.2 mM<\/td>\n<\/tr>\n
10 g\/l<\/td>\ninositol<\/td>\n55.5 mM<\/td>\n<\/tr>\n
10 mg\/l<\/td>\nbiotin<\/td>\n40.8 uM<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

<\/h4>\n

10,000x Mineral stock<\/span><\/h4>\n\n\n\n\n\n\n\n\n\n\n\n
amt<\/th>\ncomponent<\/th>\nfinal conc<\/th>\n<\/tr>\n
5 g\/l<\/td>\nboric acid<\/td>\n80.9 mM<\/td>\n<\/tr>\n
4 g\/l<\/td>\nMnSO4<\/td>\n23.7 mM<\/td>\n<\/tr>\n
4 g\/l<\/td>\nZnSO4.7H2O<\/td>\n13.9 mM<\/td>\n<\/tr>\n
2 g\/l<\/td>\nFeCl2.6H2O<\/td>\n7.40 mM<\/td>\n<\/tr>\n
0.4 g\/l<\/td>\nmolybdic acid<\/td>\n2.47 mM<\/td>\n<\/tr>\n
1 g\/l<\/td>\nKI<\/td>\n6.02 mM<\/td>\n<\/tr>\n
0.4 g\/l<\/td>\nCuSO4.5H2O<\/td>\n1.60 mM<\/td>\n<\/tr>\n
10 g\/l<\/td>\ncitric acid<\/td>\n47.6 mM<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

<\/h3>\n

MB media<\/h3>\n

This is a very stringent minimal medium that is used for lithium acetate transformation protocol<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
amt<\/th>\nCompound<\/th>\n<\/tr>\n
0.5 g\/l<\/td>\nKH2PO4<\/td>\n<\/tr>\n
0.36 g\/l<\/td>\nK-acetate<\/td>\n<\/tr>\n
0.5 g\/l<\/td>\nMgSO4.7H2O<\/td>\n<\/tr>\n
0.1 g\/l<\/td>\nNaCl<\/td>\n<\/tr>\n
0.1 g\/l<\/td>\nCaCl2.2H2O<\/td>\n<\/tr>\n
5 g\/l<\/td>\n(NH4)2SO4<\/td>\n<\/tr>\n
500 ug\/l<\/td>\nH3BO4<\/td>\n<\/tr>\n
40 ug\/l<\/td>\nCuSO4.5H2O<\/td>\n<\/tr>\n
100 ug\/l<\/td>\nKI<\/td>\n<\/tr>\n
200 ug\/l<\/td>\nFeCl3.6H2O<\/td>\n<\/tr>\n
400 ug\/l<\/td>\nMnSO4.H2O<\/td>\n<\/tr>\n
200 ug\/l<\/td>\nNa2MoO4.2H2O<\/td>\n<\/tr>\n
400 ug\/l<\/td>\nZnSO4.7H2O<\/td>\n<\/tr>\n
5g\/l<\/td>\nglucose<\/td>\n<\/tr>\n
10ug\/l<\/td>\nbiotin<\/td>\n<\/tr>\n
1mg\/l<\/td>\ncalcium pantothenate<\/td>\n<\/tr>\n
10mg\/l<\/td>\nnicotinic acid<\/td>\n<\/tr>\n
10mg\/l<\/td>\nmyo-inositol<\/td>\n<\/tr>\n
150 mg\/l<\/td>\nuracil for ura4- strains or<\/td>\n<\/tr>\n
150 mg\/l<\/td>\nleucine for leu1- strains.<\/td>\n<\/tr>\n
Filter sterilize<\/td>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Mating\/Sporulation media<\/h3>\n

Autoclave as usual<\/p>\n

ME: Malt extract<\/h4>\n\n\n\n\n\n\n
amt<\/th>\ncomponent<\/th>\nfinal conc<\/th>\n<\/tr>\n
30 g\/l<\/td>\nBacto-malt extract<\/td>\n3% (w\/v)<\/td>\n<\/tr>\n
Supplements added as for YES except lysine. Adjust to pH 5.5 with NaOH.<\/td>\n<\/tr>\n
Solid media is made by adding 2% Difco Bacto Agar<\/strong><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

<\/h4>\n

SPAS mating media<\/span><\/h4>\n\n\n\n\n\n\n\n\n
amt<\/th>\ncomponent<\/th>\nfinal conc<\/th>\n<\/tr>\n
10 g\/l<\/td>\nglucose<\/td>\n1% (w\/v)<\/td>\n<\/tr>\n
1 g\/l<\/td>\nKH2PO4<\/td>\n7.3mM<\/td>\n<\/tr>\n
1 ml<\/td>\n1000x vitimins<\/a><\/td>\n(w\/v)<\/td>\n<\/tr>\n
supplements: 45 mg\/l adenine, histidine, leucine, uracil and lysine hydrochloride (1\/5 normal).<\/td>\n<\/tr>\n
Solid media is made by adding 3% Difco Bacto Agar<\/strong><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Growth on nonfermantable carbon sources<\/h4>\n
\n

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:<\/p>\n

Strains will grow better if ura4+
\n 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<\/p>\n

 <\/p>\n

Suggested recipes<\/p>\n

\n

1% yeast extract, 0.5% casaminoacids, 3% glycerol, 3% ethanol, 40 microg\/ml adenine. (Nathalie Bonnefoy)
\n 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.
\n 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)
\n Yeast Extract -dextrose + 3% glycerol as a growth assay for S. pombe. Wild type colonies in 3-4 days. (Nate Blewitt,)
\n liquid 0.5%YE + glycerol (2%? 3%?) without any glucose. (Peter Fantes)
\n EMM doesn’t work.<\/p>\n

Thanks to Peter Fantes, Charlie Hoffman, Nate Blewitt, Nathalie Bonnefoy , and Kurt Runge for responses quoted here.<\/p>\n

\n

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