Normalized yeast two-hybrid cDNA libraries—Mate & Plate

The Normalized Mate & Plate Libraries are high-complexity cDNA libraries cloned into a GAL4 AD vector and transformed into yeast strain Y187. These libraries significantly reduce the labor and time required to perform a yeast two-hybrid screen because library amplification and yeast transformation have already been done. Additionally, normalization selectively removes highly abundant transcripts from the libraries to enhance the representations of low-abundance and rare cDNAs. This greatly reduces the possibility of obtaining false positives during screening.

Click to find additional yeast two-hybrid cDNA Mate and Plate libraries.

Is it really this easy?

Yes, there are just three simple steps:

1. Make a bait strain expressing your protein of interest using the Matchmaker Gold Yeast Two-Hybrid System.
2. Mix the bait strain overnight with a 1 ml vial of normalized library.
3. Plate on selective medium.

What is a normalized library?

Normalization reduces the proportion of highly abundant transcripts in an cDNA pool. This means that many of the most highly abundant housekeeping genes are significantly reduced in copy number so you can screen fewer clones and be assured that your screens have included medium- and low-abundance clones. In short, you can screen a greater number of independent clones with less effort, and have a greater chance of detecting important interactions.

How are normalized libraries made?

We start by generating SMART-amplified cDNA, which we then normalize using duplex-specific nuclease (DSN) normalization (Shagin et al. 2002, Zhulidov et al. 2004). Briefly, the cDNA is denatured and reannealed, followed by brief digestion with an enzyme that specifically cleaves double-stranded DNA, because the more abundant cDNAs anneal more rapidly and thus are more likely to be destroyed by the enzyme. Finally, we check the efficiency of cDNA normalization by virtual Northern blot analysis and clone the normalized cDNA library into our prey vector, pGADT7-RecAB.

Universal libraries offer universal gene coverage

Our human and mouse universal libraries provide the broadest and most complete coverage of expressed genes. These normalized, all-purpose libraries were created from a diverse collection of whole tissues that were specifically chosen to represent the most expansive range of expressed genes (Zhulidov et al. 2004). Combining “across-the-board” gene representation with the enrichment of low-copy-number cDNAs, our Mate & Plate Universal (Normalized) Libraries offer the greatest capacity for identifying novel and genuine binding partners for your protein of interest.

Why use normalized universal libraries?

Many of the most meaningful interactions in nature occur between appropriately localized, but weakly expressed proteins. These types of interactions may not be readily detectable in a traditional, single-tissue library. Universal libraries that are also normalized offer an effective solution to this problem because they represent a balanced array of transcripts covering the broadest possible range of expressed genes. Thus, your screens are not limited to identifying interactions between proteins that are highly expressed in a single tissue.