High-throughput yeast two-hybrid screening of complex cDNA libraries

Methods Mol Biol. 2012;812:89-102. doi: 10.1007/978-1-61779-455-1_5.

Abstract

Yeast two-hybrid screening can be used to find cDNAs encoding proteins which bind to a given bait protein in large, pooled cDNA libraries. Screening of complex, pooled libraries is slower and more laborious than screening of arrayed collections of cDNAs, but has several advantages. First, the complexity of a pooled library can be orders of magnitude larger than the size of a typical arrayed library. Second, as long as available cDNA collections are incomplete and limited to full-length cDNAs, pooled cDNA libraries offer a more complete search space and are often the only way to do screens in organisms other than human and a few model organisms. We have streamlined and optimised the screening of pooled libraries in a format which uses micro-titre plates and produces quantitative signals for the selection of hits. This format has the advantage that automation of the process is straightforward and allows a throughput of up to at least 1,000 screens per year per person.

MeSH terms

  • Animals
  • Gene Library*
  • Mice
  • Polymerase Chain Reaction
  • Transformation, Genetic
  • Two-Hybrid System Techniques*
  • Yeasts / cytology
  • Yeasts / genetics