The Yeast Protocols Handbook provides background information and general yeast protocols that

complement our system-specific User Manuals. The protocols in this Handbook have been optimized

with our yeast-based Matchmaker™ Two-Hybrid and One-Hybrid Systems, and Matchmaker

Libraries. The Yeast Protocols Handbook is especially useful for researchers who wish to use

yeast as a vehicle for their molecular biology experiments, but have little or no prior experience

working with yeast. For novice and experienced users alike, the Yeast Protocols Handbook will

help you obtain the best possible results with your Matchmaker and other yeast-related products

from Clontech.

This Handbook includes:

• detailed information on culturing and handling yeast

• information on the yeast promoters used in the Matchmaker Systems

• two protocols for preparing protein extracts from yeast

• quantitative and qualitative β-galactosidase assays (for use with lacZ yeast reporter

strains)

• a simple, optimized protocol for isolating plasmids from yeast

• PCR amplification and yeast colony hybridization protocols for the rapid analysis of positive

clones obtained in a library screening

• a small-scale, lithium acetate yeast transformation protocol

• additional protocols for working with certain yeast plasmids and host strains

The special application of yeast transformation for one- and two-hybrid library screening is covered

in detail in each product-specific User Manual. The special application of yeast mating for library

screening is covered in the Pretransformed Matchmaker Libraries User Manual.

About our yeast-based products

The Matchmaker GAL4 Two-Hybrid Systems (Cat No. K1604-1, K1605-1, 630303) and LexA TwoHybrid System (Cat No. K1609-1) are complete kits for identifying and investigating protein-protein

interactions in vivo using the yeast two-hybrid assay. The Matchmaker One-Hybrid System (Cat

No. K1603-1) provides the basic tools for identifying novel proteins in vivo that bind to a target

DNA sequence such as a cis-acting regulatory element. Matchmaker Two-Hybrid Systems are

compatible with our pBridge Three-Hybrid Vector (Cat No. 630404) for the investigation of tertiary

protein complexes. The Matchmaker Libraries are constructed in vectors that express inserts as

fusions to a transcriptional activation domain, and are thus a convenient resource for researchers

wishing to screen a library using the one- or two-hybrid assays. Pretransformed Matchmaker

Libraries provide an even greater level of convenience for those wishing to perform a two-hybrid

library screening without using large- or library-scale yeast transformations.

Clontech offers an extensive line of kits and reagents that support and complement the

Matchmaker Systems and Libraries. The YeastmakerTMYeast Transformation Kit (Cat No. 630439)

includes all the necessary reagents and protocols for efficient transformation using the lithium

acetate method. Also available from Clontech: a selection of GAL4 DNA-binding domain (DNA-BD)

and activation domain (AD) hybrid cloning vectors; the pGilda Vector for use with LexA-based

two-hybrid systems; monoclonal antibodies and sequencing primers; and yeast media, including

Minimal SD Base and many different formulations of Dropout (DO) Supplement. Finally, the pHACMV and pMyc-CMV Vector Set (Cat No. 631604) can be used to confirm protein interactions in

mammalian cells.

For ordering information on these products, please see Chapter XI of this Handbook or the

Clontech Catalog

LacZ is a large gene, which may complicate subsequent clonging and enzymatic manipulation steps. Can't you try GFP? It's not only smaller, but easier to quantify.