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Overview
The Yeast GFP (Green Fluorescent Protein) Clone Collection of Saccharomyces cerevisiae tagged open reading frames (ORF) were generated by Dr. Erin O’Shea and Dr. Jonathan Weissman at University of California-San Francisco (Huh et al., 2003).The strategy for constructing the Yeast GFP Clone Collection is described below.
The Yeast GFP Clone Collection is a S. cerevisiae yeast strain collection expressing full-length ORFs containing an Aequorea victoria GFP (S65T) tag (Tsien, 1998) at the C-terminus end. The GFP fusion proteins are integrated into the yeast chromosome through homologous recombination and are expressed using endogenous promoters. The Yeast GFP Clone Collection consists of 4,159 GFP tagged ORFs comprising 75% of the yeast proteome. The GFP fusion proteins were localized to 22 distinct subcellular locations using fluorescence microscopy.
Applications
The Yeast GFP Clone Collection can be used for the following applications:
Strategy
The strategy used to generate the Yeast GFP Clone Collection is described below.
Figure 1
Advantages
Using the Yeast GFP Clones for localization studies offers the following advantages:
Replacements
As a distributor of Yeast GFP Clone Collection, Thermo Fisher Scientific cannot be held responsible for errors in genotype phenotype, and localization. Replacements are issued at the discretion of Thermo Fisher Scientific.
References
Introduction
Instructions for preparing glycerol stocks and handling plates upon receipt of the Yeast GFP Clones are described below. Protocols for using the Yeast GFP Clones in specific applications are not included in this manual.
Preparing glycerol stocks
Prepare a glycerol stock culture within 2 weeks of receiving the clone stab.
Inoculation from plates
Allow the plate to thaw completely and resuspend the cells before replication as yeast has a tendency to settle to the bottom (flocculate) of the well during growth. Avoid freeze-thaw as it decreases cell viability.
Avoiding cross contamination
YPD medium
Yeast Extract Peptone Dextrose Medium (1 liter)
1% yeast extract
2% peptone
2% dextrose (D-glucose)
Note: If making plates, omit dextrose from Step 1. Autoclaving agar and dextrose together will cause the
dextrose to caramelize. Prepare a separate stock solution of 20% dextrose and autoclave or filter-sterilize. After the YPD broth (900 mL volume) has been autoclaved, add 100 mL of 20% dextrose to the medium.