Gene identification using the yeast two-hybrid system

Methods in Enzymology

Volumn 283, Issue , 1997, Pages 141-156

  Bai, Chang ^a   Elledge, Stephen J ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; CLONING VECTOR; CONTROLLED STUDY; GENE ISOLATION; HYBRIDIZATION; NONHUMAN; PRIORITY JOURNAL; PROTEIN DNA BINDING; PROTEIN PROTEIN INTERACTION;

EID: 0030839829     PISSN: 00766879     EISSN: None     Source Type: Book Series    
DOI: 10.1016/S0076-6879(97)83013-3     Document Type: Article

References (23)

1. S. Fields and O. Song, Nature (London) 340, 245 (1989).
2. C.-T. Chien, P. L. Bartel, R. Sternglanz, and S. Fields, Proc. Natl. Acad. Sci. U.S.A. 88, 9578 (1991).
3. B. Li and S. Fields, FASEB J. 7, 957 (1993).
4. J. W. Harper, G. R. Adami, N. Wei, K. Keyomarsi, and S. J. Elledge, Cell 75, 805 (1993).
5. H. Toyoshima and T. Hunter, Cell 78, 67 (1994).
6. S. Fields, Trends Genet. 10, 286 (1994).
7. J. Gyuris, E. Golemis, H. Chertkov, and R. Brent, Cell 75, 791 (1993).
8. F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (eds.), "Current Protocols in Molecular Biology." Wiley, New York, 1994.
9. C. Guthrie and G. R. Fink, Methods Enzymol. 194 (1991).
10. T. Durfee, K. Becherer, P. L. Chen, S. H. Yeh, Y. Yang, A. E. Kilburn, W. H. Lee, and S. J. Elledge, Genes Dev. 7, 555 (1993).
11. P. Bartel, C.-T. Chien, R. Sternglanz, and S. Fields, Biotechniques 14, 920 (1993).
12. S. Fields, Methods 5, 116 (1993).
13. J. T. Mulligan and S. J. Elledge, in "Molecular Genetics of Yeast: A Practical Approach," p. 65. Oxford University Press, Oxford, 1994.
14. PEG can be briefly heated in a microwave oven to help it go into solution if necessary.
15. L. Breeden and K. Nasmyth, Cold Spring Harbor Symp. Quant. Biol. 50, 643 (1985).
16. We observe that nitrocellulose filter becomes more brittle in liquid nitrogen than do nytran filters. However, nitrocellulose filters adhere to colonies slightly better than does nytran. Filters can be thoroughly washed with deionized water and reused.
17. It is best to have at least medium-size colonies with which to perform this assay.
18. R. D. Gietz and R. A. Woods, in "Molecular Genetics of yeast: A Practical Approach." Oxford University Press, Oxford, 1994.
19. K. Kohrer and H. Domdey, Methods Enzymol 194, 398 (1991).
20. SC-Trp medium is used to select for pAS-X but YPED gives the best transformation efficiencies. The doubling time for Y190 (pAS-X) in YPED varies between 2.0 and 3.5 hr depending on the plasmid it carries.
21. We have found that total yeast RNA works more reproducibly as a carrier but more work is required to prepare than DNA. Protocols for preparing ssDNA and yeast RNA for transformation can be found in Refs. 15 and 16, respectively.
22. Cells in PEG are more fragile and often die when pelleted; thus the recovery step is useful. Plating directly from the PET without the recovery step also works, but is more messy. Cells lose less than half their viability when frozen and can be stored indefinitely. This is useful because they can be thawed and plated at a optimal density for screening/selection at leisure.
23. T. H. Lee, S. J. Elledge, and J. S. Butel, J. Virol. 69, 1107 (1995).