Media and growth conditions

We routinely culture yeast in YPD-rich medium. Once a plasmid is introduced, yeast should be grown in synthetic drop-out medium to select for the plasmid. For example, after transforming YEp51-Bax into yeast, transformants are initially selected and subsequently maintained on synthetic drop-out medium lacking leucine (SD-Leu) and supplemented with glucose to repress Bax expression. We observed that the amount of nutrients in the medium can also affect the sensitivity of yeast to pro-apoptotic proteins. In general, we find that yeast are more sensitive to Bax when cultured in medium with minimal nutrients. For this reason, Burkholder's Minimal Medium (BMM) medium (71) may be useful for detecting a more striking lethal effect of Bax in yeast. The preparation of commonly used media is listed below (see refs 72 and 73 for detailed descriptions of S. cerevisiae media).

Protocol 30. YPD medium Method

1. To a 2 litre flask, add 10 g Bacto-yeast extract and 20 g Bacto-peptone. Include 20 g of agar when making YPD plates.

2. Bring the volume to 900 ml with distilled H20.

3. Following autoclaving, add 100 ml of filter-sterilized 20% glucose (dextrose) stock.

4. When making plates, let the medium cool to 65°C and then pour into Petri dishes. 1 litre of medium should yield about 50 100 mm diameter plates.

Protocol 31. SD medium

There are three steps to the preparation of SD medium.

Method

1. Prepare the following amino acid and supplement stocks in distilled H20: 4 mg/ml each of adenine, l-histidine-HCI, l-arginine-HCI, l-methionine, l-leucine, l-isoleucine, l-lysine-HCI, phenylalanine, aspartic acid, and valine; 2 mg/ml of uracil. Sterilize by autoclaving. 4 mg/ml each of l-tryptophan, l-tyrosine, and l-threonine and sterilize by filtration. Store at room temperature.

2. To a 2-litre Erlenmeyer flask, add 6.7 g yeast nitrogen base without amino acids, and 10 ml each of the above amino acid stocks and 20 ml of uracil, leaving out the three heat-sensitive amino acids (tryptophan, tyrosine, and threonine). Omit the amino acid(s) that will be used for selection of the plasmid(s). Add 20 g of agar when making solid medium. Bring the volume to 870 ml with distilled H20 and autoclave.

3. Cool the solution to about 80°C, then add 10 ml each of the three heat-sensitive amino acids and 100 ml of filter-sterilized sugar stocks (either 20% glucose or 20% galactose; we often supplement galactose medium with 1% raffinose). SD plates are prepared similarly as described for YPD plates above.

Protocol 32. BMM medium Method

1. Prepare the following stock solutions in distilled H20. Mineral stocks (10000 X)

(a) To a 100-ml Erlenmeyer flask, add 30 mg H3B03, 50 mg MnS04.7H20, 150 mg ZnS04.7H20, and 20 mg CaS04.5 H20. Bring the volume to 50 ml with dH20.

(b) To a 100-ml Erlenmeyer flask, add 100 mg Na2Mo04.2H20. Bring the volume to 50 ml with dH20.

(c) To a 100-ml Erlenmeyer flask, add 125 mg FeCI3.6H20. Bring the volume to 50 ml with dH20.

(d) Do not autoclave. Store at 4°C. Vitamin stock (1000 x)

To a 250-ml Erlenmeyer flask, add 20 mg thiamine, 20 mg pyridoxine, 20 mg nicotinic acid, 20 mg pantothenic acid, 0.2 mg biotin, and 1 g inositol, add H20 to 100 ml. Filter sterilize and store at 4°C.

2. To a 2-litre Erlenmeyer flask, add 1.5 g KH2P04, 500 mg MgS04.7H20, 330 mg CaCI2.2H20, 100 |i,g Kl, 2 g asparagine. 100 |xl each of mineral stocks (a), (b), and (c) (10000 x) and 10 ml each required amino acids (the ones that yeast can not make) except the heat-sensitive ones (amino acid stocks are made as described for SD medium). Include 20 g agar when making solid medium. Bring the volume to 900 ml with dH20 and autoclave.

3. Following autoclaving and cooling to about 80°C, add 1 ml vitamin stock (1000 x), 10 ml each of heat-sensitive amino acids and 100 ml of appropriate carbon source (from 20% glucose or galactose stock as described for SD medium). BMM plates are made similarly as described for YPD or SD plates.

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