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Plant Gene Regulatory Networks pp 47–65Cite as

Yeast One- and Two-Hybrid High-Throughput Screenings Using Arrayed Libraries

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1629))

Abstract

Since their original description more than 25 years ago, the yeast one- and two-hybrid systems (Y1H/Y2H) have been used by many laboratories to detect DNA–protein (Y1H) and protein–protein interactions (Y2H). These systems use yeast cells (Saccharomyces cerevisiae) as a eukaryotic “test tube” and are amenable for most labs in the world. The development of highly efficient cloning methods has fostered the generation of large collections of open reading frames (ORFs) for several organisms that have been used for yeast screenings. Here, we describe a simple mating based method for high-throughput screenings of arrayed ORF libraries with DNA (Y1H) or protein (Y2H) baits not requiring robotics. One person can easily carry out this protocol in approximately 10 h of labor spread over 5 days. It can also be scaled down to test one-to-one (few) interactions, scaled up (i.e., robotization) and is compatible with several library formats (i.e., 96, 384-well microtiter plates).

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Acknowledgments

The work in L.O.-S. lab is supported by MINECO grants BIO2013-46076-R and BIO2016-77840-R. We thank all people that contributed to the development of the Arabidopsis TF library [22] as well as all the labs that have used this resource helping this way to ascertain its potential. Dr. Begoña Benito is also thanked for critical reading. We apologize to those publications not cited here due to space limitations.

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Authors and Affiliations

  1. Centro de Biotecnología y Genómica de Plantas (UPM-INIA), Universidad Politécnica de Madrid, Campus de Montegancedo, Pozuelo de Alarcón, 28223, Madrid, Spain

    Rocío Sánchez-Montesino & Luis Oñate-Sánchez

Authors
  1. Rocío Sánchez-Montesino
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  2. Luis Oñate-Sánchez
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Correspondence to Luis Oñate-Sánchez .

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Editors and Affiliations

  1. Department for Plant Cell and Molecular Biology, Institute for Biology, Humboldt-Universität zu Berlin, Berlin, Germany

    Kerstin Kaufmann

  2. Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany

    Bernd Mueller-Roeber

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Sánchez-Montesino, R., Oñate-Sánchez, L. (2017). Yeast One- and Two-Hybrid High-Throughput Screenings Using Arrayed Libraries. In: Kaufmann, K., Mueller-Roeber, B. (eds) Plant Gene Regulatory Networks. Methods in Molecular Biology, vol 1629. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7125-1_5

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  • DOI: https://doi.org/10.1007/978-1-4939-7125-1_5

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7124-4

  • Online ISBN: 978-1-4939-7125-1

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