Yeast Nitrogen Base

Yeast strains are grown on either standard yeast extract, peptone, glucose media (YPD) (1% (w/v) yeast extract, 2% (w/v) bactopeptone, and 2% (w/v) glucose) and supplemented with the appropriate antibiotic, or in synthetic complete media (SCD media) (0.17% (w/v) yeast nitrogen base. 0.5% (w/v) ammonium sulphate, 2% (w/v) glucose, and supplemented with 20 mg/1 arginine, 100 mg/1 aspartic acid, 100 mg/1 glutamine, 30 mg/1 isoleucine, 30 mg/llysine, 20 mg/1 methionine, 50 mg/1 phenylalanine, 400 mg/lserine, 200 mg/1 threonine, 30 mg/1 tyrosine, and 150 mg/1 valine. When needed, the media was also supplemented with 20 mg/1 adenine, 10 mg/1 leucine, 60 mg/1 histidine, 60 mg/1 tryptophan, and 20 mg/1 uracil). 

Cells (C), or supernatant liquor (S). b YCV, Yeast nitrogen base-Casamino acids-Vitamins. M, Synthetic medium. d BHI, Brain-heart infusion. Sab, Sabouraud.1 B, Mariat s medium. 

Yeast Genetics. The yeast strain used in these studies was YGXD8 (MAT a /ew2-3 leu2-112). Yeast cells were transformed by the spheroplast method of Hinnen et al. (21). The transformed cells were maintained in YNB medium (0.7% yeast nitrogen base) supplemented with 5% glucose. For induction of polyphenolic protein synthesis, the cells were cultured in YP medium (1% yeast extract, 1% bacto-peptone) supplemented with 4% glucose and 2% galactose. 

Yeast Nitrogen Base Dextrose (YNBD) Medium... 

Yeast nitrogen base without amino acids and 1.72... 

Synthetic dropout medium can either be purchased readymade (for example from Sigma-Aldrich, Clontech or MP Biomedicals) or made from individual components ( eeNote 5). Synthetic dropout medium is comprised of 8 g yeast nitrogen base, 55 mg adenine sulfate, 55 mg tyrosine, 55 mg uracil (for Ura strains), and 20 g glucose per liter. Synthetic dropout plates contain, in addition, 22 g agar per liter. A lOOx concentrated amino acid dropout stock contains 2 g arginine, 1 g histidine, 6 g isoleucine, 6 g leucine, 4 g lysine 1 g methionine, 6 g phenylalanine, 5 g threonine, and 4 g tryptophan per liter. Amino acids necessary for selection are omitted. Synthetic complete (SC) medium has no amino acids omitted from the dropout. 

Following suppliers instructions, prepare yeast nitrogen base (YNB) and agar at 2% (wt/vol). Dissolve in a boiling waterbath and dispense 15-20 mL aliquotes into stoppered test tubes and autoclave per recommendations. 

Assimilation tests for carbon compounds use yeast nitrogen base (YNB) without carbon sources, and the assimilation test for nitrogen compounds use yeast carbon base (YCB) without assimilable nitrogen sources. For the tests described, YNB without amine acids and ammonium sulfate is used (Anonymous, 1984). Carbon sources normally examined include a number of pentoses, hexoses, disaccharides, trisaccharides, polysaccharides, alcohols, organic acids, and glycosides as specified by Yarrow (1998). Nitrogen sources commonly tested include nitrate, nitrite, ethyl-amine hydrochloride, cada-verine dihydrochloride, L-lysine, imidazole, glucosamine, creatine, and creatinine. When nitrite is used as a test compound, it is necessary to adjust the pH of the medium to 6.5 because toxic nitrous acid is formed at pH <6.0. 

Complete minimal (CM) dropout plates 0.17% (w/v) yeast nitrogen base, 0.5% (w/v) ammonium sulfate, 2% (w/v) dextrose, 2% (w/v) bacto agar, complete supplemental mixture. 

Transformation selection medium synthetic dextrose (SD) dropout media (0.17% yeast nitrogen base without amino acids and without ammonium sulfate, 0.5% ammonium sulfate, 2% glucose, and required nutrients) (1). 

SC-Ura For 1 L of medium, add 6.7 g of yeast nitrogen base w/o amino acids, 20 g of dextrose, and 1.92 g of yeast synthetic drop-out medium supplement without uracil. Dissolve in ddH O, adjusting the volume to 1 L, and autoclave. 

Synthetic media can be prepared in two ways. The most convenient, although expensive, way is to use Bacto yeast nitrogen base without amino acids as supplied by Difco Laboratories (Detroit, Mich., No. 0919-15). However, a medium of equivalent quality can be composed according to the following formula, beginning with a list of ingredients for a stock solution ... 

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