Clontech’s Matchmaker Gold Yeast One-Hybrid (Y1H) Library Screening System provides a simple and efficient method for identifying and characterizing novel protein-DNA interactions. All Matchmaker Gold Systems use Aureobasidin A resistance (AbAr) as a stringent, highly selective, and easy-to-use reporter. This novel reporter produces very low screening backgrounds, since the Aureobasidin A antibiotic (AbA) efficiently kills yeast lacking AbAr expression.
The Matchmaker Gold Yeast One-Hybrid System
In the Matchmaker Gold Yeast One-Hybrid System, tandem repeats of your DNA target/bait sequence are cloned into the pAbAi reporter vector. To generate your bait-specific reporter strain, the pAbAi vector is then integrated into the genome of Y1HGold yeast using homologous recombination. This strain provides a host for library screening.
One-Step Library Construction and Screening
A cDNA library of potential DNA-binding proteins, which are ultimately expressed as fusions to the yeast GAL4 transcription activation domain (GAL4 AD prey proteins), is constructed directly in the Y1HGold[Bait-AbAi] reporter strain using SMART technology and homologous recombination. When a prey protein binds to the bait sequence, its associated GAL4 AD activates AbAr expression, allowing the cell to grow on medium containing AbA. In library screens, the plasmids encoding the library-derived prey proteins can be easily rescued from the surviving yeast clones and subjected to further analysis.
Get Results Fast!
With Matchmaker Gold, one-hybrid library screening is straightforward, quick and easy:
Step 1: Create a bait construct by cloning 1–3 tandem repeats of the target DNA-binding sequence into pAbAi.
Step 2: Create a bait-specific reporter strain by the transforming and integrating the linearized pBait-AbAi construct into the Y1H Gold yeast strain and selecting on SD/–Ura agar medium.
Step 3: Confirm the integration of the bait sequence using colony PCR and the Matchmaker Insert Check PCR Mix 1.
Step 4: Use SMART technology to synthesize cDNA containing ends that are homologous to the ends of the linearized pGADT7-Rec vector.
Step 5: Create and screen your library in a single step by cotransforming your bait-specific reporter strain with the SMART-generated cDNA and the linearized pGADT7-Rec vector, and plating on AbA-containing selective medium.
Step 6: Harvest the resulting colonies, which contain putative DNA-binding prey proteins, and analyze your library inserts using the Matchmaker Insert Check PCR Mix 2.
Analysis by Colony PCR
Screening positive colonies by PCR is a fast and convenient way to analyze the bait strain and sort through the positive clones identified in Y1H and Y2H screens. With the Matchmaker Insert Check PCR Mix 1, you can verify the integration of your Y1H pBait-AbAi construct and with the companion Matchmaker Insert Check PCR Mix 2, you can quickly sort through and analyze the inserts in positive clones that have emerged from either one-hybrid or two-hybrid screens.
Yeast One-Hybrid Media
Our Yeast One-Hybrid Media Sets each contain a complete set of the all the Yeast Media Pouches you need for Clontech’s Matchmaker Gold One-Hybrid protocols.