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Growth and Manipulation of Yeast

Douglas A. Treco¹, Ann Reynolds², Victoria Lundblad³

¹Massachusetts General Hospital, Boston, Massachusetts
²University of Washington, Seattle, Washington
³Baylor College of Medicine, Houston, Texas

Publication Name:

Current Protocols in Protein Science

Unit Number:

Appendix 4L

DOI:

10.1002/0471140864.psa04ls14

Online Posting Date:

May, 2001

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Abstract

This unit describes preparation of selected media for growing yeast and also discusses strain storage and revival. Protocols are provided for the assay of -galactosidase in liquid culture and for transformation using lithium acetate.

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Preparation of Selected Yeast Media
Strain Storage and Revival
Reagents and Solutions
Literature Cited
Tables

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Materials

Basic Protocol 1: Assay for -Galactosidase in Liquid Cultures

Materials

YPD medium (see recipe under Liquid Media)
Z buffer (see recipe in Reagents and Solutions)
0.1% sodium dodecyl sulfate(SDS)
Chloroform
4 mg/ml O-nitrophenyl--d-galactoside (ONPG) in 0.1 M potassium phosphate, pH 7.0 (see appendix 2E for buffer; filter sterilize and store frozen)
1 M Na₂CO₃
30°C water bath

Basic Protocol 2: Transformation Using Lithium Acetate

Materials

YPD medium (see recipe under Liquid Media)
Yeast strain to be transformed
YPAD medium: YPD medium supplemented with 30 mg/liter adenine hemisulfate
Highest-quality sterile H₂O
10× TE buffer, pH 7.5 (see appendix 2E for 1× recipe), sterile
10× lithium acetate stock solution: 1 M lithium acetate, pH 7.5 (adjust pH with dilute acetic acid), filter sterilized
DNA: high-molecular-weight, single-stranded carrier DNA and transforming DNA
50% (w/v) polyethylene glycol (PEG 4000 or 3350 (do not use PEG 8000), filter sterilized
CM dropout plates (see recipe under Solid Media) prepared with Difco agar
30°C incubator with shaker
Sorvall GSA and SS-34 rotors (or equivalents)
42°C water bath