Yeast two-hybrid (Y2H) systems are primarily used for screening a complete library of proteins (prey) for interaction with a specific protein of interest (bait). However, the manufacturing and screening of these libraries has traditionally been both time- and labor-intensive. To address these shortcomings, we've developed our popular, ready-to-go Mate & Plate libraries that make Y2H screens simple: co-culture your library strain with your bait strain, plate on the appropriate selective minimal media, and you're done.

If our selection of ready-made libraries doesn't suit your needs, we offer the Make Your Own "Mate & Plate" Library System. This kit provides the necessary materials and a simple, highly efficient method to make enough Mate & Plate library for hundreds of Y2H screens, all in less than a week.

Library creation occurs directly in our library Y187 Yeast Strain, utilizing the highly efficient homologous recombination machinery of S. cerevisiae (Figure 1, Panel A). This system uses our proprietary SMART cDNA synthesis technology (Figure 1, Panel B), allowing you to construct cDNA libraries from any tissue source with as little as 100 ng of total input RNA. This eliminates the need for the labor-intensive library cloning, amplification, and harvesting in E. coli required in traditional library construction methods.

The highly efficient homologous recombination pathways of S. cerevisiae yeast have been well-documented (Pâques and Haber 1999; Sung et. al. 2000). Their efficiency has been exploited for decades to enable E. coli-free cloning of plasmids through a process known as gap-repair (Ma et al. 1987). We've taken this one step further by providing a way for you to perform E. coli-free cloning of an entire library directly in yeast. The entire process consists of four simple steps:

Step 1: first-strand synthesis using the included SMART oligo to generate cDNA homologous at both ends to pGADT7-Rec
Step 2: second-strand PCR synthesis to generate 2–5 µg of library cDNA
Step 3: cotransformation and recombination in the Y187 Yeast Strain
Step 4: harvesting, mixing, and aliquoting of colonies into 1-ml single-use vials, providing enough material for hundreds of Y2H screens

The cDNA size range for inserts cloned with this protocol is 0.3–6 kb (Figure 2). As the SMART oligo sequence is required for homology to the prey vector, prematurely terminated reverse transcripts are selected against because they cannot be amplified using the second-strand synthesis primers. Our Make Your Own "Mate & Plate" Library System also contains CHROMA SPIN gel filtration columns to size-select for larger (>400 bp) cDNAs (Figure 2) and deliver complex, representative libraries (Figure 3).

Ma, H., Kunes, S., Schatz, P. J. & Botstein, D. Plasmid construction by homologous recombination in yeast. Gene 58, 201–16 (1987).

Pâques, F. & Haber, J. E. Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 63, 349–404 (1999). 

Sung, P., Trujillo, K. M. & Van Komen, S. Recombination factors of Saccharomyces cerevisiae. Mutat. Res. 451, 257–75 (2000).