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Growth and Manipulation of Yeast
Publication Name:
Current Protocols in Molecular Biology
Unit Number:
Unit 13.2
DOI:
10.1002/0471142727.mb1302s82
Online Posting Date:
April, 2008 Abstract
Yeast cultures can be grown, maintained, and stored in liquid media or on agar plates using techniques similar to those for bacterial cultures. This unit describes culture conditions for these basic techniques. Additional methods describe determination of yeast mating type, diploid construction, sporulation, tetrad dissection, and random spore analysis. Curr. Protoc. Mol. Biol. 82:13.2.1-13.2.12. © 2008 by John Wiley & Sons, Inc.
Keywords: yeast; media; yeast culture; growth
Table of Contents
- Introduction
- Basic Protocol 1: Growth in Liquid Media
- Basic Protocol 2: Growth on Solid Media
- Basic Protocol 3: Determination of Cell Density
- Basic Protocol 4: Determination of Phenotype by Replica Plating
- Basic Protocol 5: Determination of Mating Type
- Basic Protocol 6: Diploid Construction
- Basic Protocol 7: Sporulation of Diploid Cells
- Basic Protocol 8: Preparation and Dissection of Tetrads
- Support Protocol: Preparation of Dissecting Needles
- Alternate Protocol: Random Spore Analysis
- Commentary
- Bibliography
- Figures
Materials
Basic Protocol 5: Determination of Mating Type
Materials
- S. cerevisiae: MAT thr4
– (tester), MAT thr4– (tester), and uncharacterized strains - YPD medium and plates (unit
- Minimal plates (unit
- Replica plating block (unit
- Sterile velvets (unit
Basic Protocol 7: Sporulation of Diploid Cells
Materials
- Yeast cells
- Sporulation plates or sporulation medium, with appropriate nutrients (unit
- YPD medium (unit
Basic Protocol 8: Preparation and Dissection of Tetrads
Materials
- Spores (see
- 0.5 mg/ml Zymolyase-100T (ICN Immunobiologicals) in 1 M sorbitol
- Dissecting microscope
- Dissecting needle (see Support Protocol)
Alternate Protocol: Random Spore Analysis
Materials
- Spores (see
- 1 mg/ml Zymolyase-20T (ICN Immunobiologicals) in H
2 O, filter sterilized - 2-Mercaptoethanol (2-ME)
- 1.5% (v/v) Nonidet P-40 (NP-40)
- Ethanol
- Sonicator and probe
- Additional reagents and equipment for cell counting (unit
Looking for materials? Suppliers Guide
Figures
-
Figure 13.2.1Essential features of the dissection microscope. Not all parts are shown, including the light source and condenser (below) and the objective and eyepiece (above). The micromanipulator is attached to the base of the microscope. The mark with the large dot on the y-axis guide is aligned with demarcations on the right side of the stage to identify positions a, b, c, and d (corresponding to positions 10, 15, 20, and 25 respectively, in this figure). The upper right-hand corner of the plate holder is aligned with demarcation along the rear of the stage to identify positions 1, 213 (corresponding to positions 60, 65120, respectively in this figure). -
Figure 13.2.2Preparation of glass needles for tetrad dissection.
Literature Cited
| Literature Cited | |
| Esposito, R.E. and Klapholz, S. 1981. "Meiosis and ascosporal development". In The Molecular Biology of Yeast Saccharomyces: Life Cycle and Inheritance ( J.N. Strathern, E.W. Jones, and J.R. Broach, eds.) pp. 211-287. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. | |
| Sherman, F. 2002. "Getting started with yeast". In Guide to Yeast Genetics and Molecular and Cell Biology, Part B (C. Guthrie and G.R. Fink, eds.) pp. 3-41. Academic Press, San Diego. | |
| Wang, H-T., Frackman, S., Kowalisyn, J., Esposito, R.E., and Elder, R. 1987. Developmental regulation of SPO13, a gene required for separation of homologous chromosomes at meiosis I. Mol. Cell. Biol. 7: 1425-1435. | |
| Key Reference | |
| Sherman, F., Fink, G.R., and Lawrence, C.W. 1979. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. | |
| Provides a number of detailed procedures for genetic experiments that may be of interest to more advanced students. | |
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