Applications of Chimeric Genes and Hybrid Proteins - Part C: by Melvin I. Simon, Jeremy Thorner, Scott D. Emr, John N.
By Melvin I. Simon, Jeremy Thorner, Scott D. Emr, John N. Abelson
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Extra resources for Applications of Chimeric Genes and Hybrid Proteins - Part C: Protein-Protein Interactions and Genomics
Control experiments should be performed to demonstrate that the Bait protein is stable, can enter the nucleus and bind lexA operator sites, and does 6 S. M. HoUenberg, R. Sternglanz, P. F. Cheng, and H. Weintraub, MoL Cell. BioL 15, 3813 (1995).  LexA-BASED TWO-HYBRID SYSTEMS 19 not appreciably activate transcription of the lexA operator-based reporter genes. Results from these control experiments will dictate if the Bait can be used to screen a library, using the initial test conditions, or if modifications of the Bait and/or different combinations of reporter strains/plasmids must be used.
Winston, and P. " Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1990. 10 TWO-HYBRID METHODS AND RELATED TECHNIQUES  pendent colonies per transformation for each array element. The pooling of too many colonies risks the possibility of variants outcompeting the desired one in culture. We determined experimentally that the sum of all errors introduced in the cloning process was approximately 10% (oligonucleotide synthesis, PCR, recombination cloning) and reasoned that the presence of two independent colonies would increase the probability that at least one correctly synthesized fusion protein would be present per array element.
Golemis and I. Serebriiskii, in "Cells: A Laboratory Manual" (D. L. Spector, R. Goldman, and L. ). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1997. 9 M. A. Watson, R. Buckholz, and M. P. Weiner, BioTechniques 21, 255 (1996). 10D. Gietz, A. St. Jean, R. A. Woods, and R. H. Schiestl, Nucleic Acids Res. 20, 1425 (1992). 11 j. Hill, K. A. Ian, G. Donald, and D. E. Griffiths, Nucleic Acids Res. 19, 5791 (1991). ] can vary among colonies, thereby producing an apparent (and misleading) heterogeneity in Bait-mediated transcriptional activation of the two reporters.