Protocol abstract
Nature Protocols 1, - 569 - 576 (2006)
Published online: 27 June 2006 | doi:10.1038/nprot.2006.80
Subject Categories: Computational and theoretical biology | Genomics and proteomics | Model organisms | Pharmacology and toxicology
A microarray-based protocol for monitoring the growth of yeast overexpression strains
Rebecca A Butcher1,2,3 & Stuart L Schreiber1,2
Abstract
Gene overexpression can be used to investigate the biological pathways that are important in the response to a small molecule or other perturbation. To facilitate the use of gene overexpression in the study of small-molecule mechanisms, we developed a microarray-based protocol for monitoring the growth of a pool of yeast strains, each overexpressing a different protein. In this protocol, yeast harboring a set of 
3,900 galactose-inducible overexpression plasmids are grown in the absence or presence of a small molecule for multiple generations. The plasmids are then extracted from the two populations, processed and labeled in such a manner that their relative concentrations can be determined by competitive hybridization to a microarray. Although this protocol was developed for monitoring a specific set of overexpression plasmids, it could presumably be adapted to monitor yeast that have been transformed with any set of plasmids for which the gene inserts have been spotted, or otherwise arrayed, in a microarray format. This protocol can be completed in approximately 15 hours of hands-on time over the course of several days.
- Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.
- Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA.
- Present Address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
Correspondence to: Rebecca A Butcher1,2,3 e-mail: rebecca_butcher@hms.harvard.edu
Correspondence to: Stuart L Schreiber1,2 e-mail: stuart_schreiber@harvard.edu
nature-products
A-Z product listing
- 96-well thermocycler with hot bonnet(MJ Research)
- Aminoallyl dUTP(Sigma)
- Chloroform/isoaamyl alcohol(Sigma)
- Cy3 Mono-Reactive Dye Pack, Cy5 Mono-Reactive Dye Pack(Amersham Biosciences)
- DNase-free water, RNase-free water(Ambion)
- Domed 0.2 ml microtubes(VWR)
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