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288 Tikhmyanova et al.the polylinker in the pJG4-5 vector makes it difficult to design high-qualityprimers in this region.31. Gel analysis produces little information on the completeness of digestion, becauseit is not possible to distinguish between plasmid species cut by one and twoenzymes. Purification of the digested plasmid is not necessary.32. This control experiment is an indicator of the degree of digestion of the libraryplasmid. The background level of colonies transformed with digested emptylibrary plasmid (a) (see Subheading 3.3.6.1., step 11) should be minimal. In casethe background is high, make sure that the digestion of the empty library plasmidis full and not partial by increasing the digestion incubation time or the restrictionenzyme concentration.33. The fraction of the correct clones can be assessed by replica-plating 12–24 clonesto check their phenotype (as in Subheading 3.3.5.). Normally, it should bebetween 85 and 95%.34. When transformed together, the PCR-amplified cDNA fragment from pPreycontrolPCR product and the digested library plasmid will undergo homologousrecombination in vivo in up to 97% of the transformants that acquired both vectorand insert (25,26). This is owing to the identity between the cDNA PCR fragmentand the plasmid at the priming sites. The background level of colonies transformedwith digested empty library plasmid (a) should be minimal.References1. Fields, S. and Song, O. (1989) A novel genetic system to detect protein-proteininteraction. Nature 340, 245, 246.2. Rual, J. F., Venkatesan, K., Hao, T., et al. (2005) Towards a proteome-scale mapof the human protein-protein interaction network. Nature 437, 1173–1178.3. Stelzl, U., Worm, U., Lalowski, M., et al. (2005) A human protein-protein interactionnetwork: a resource for annotating the proteome. Cell 122, 957–968.4. Giot, L., Bader, J. S., Brouwer, C., et al. (2003) A protein interaction map ofDrosophila melanogaster. Science 302, 1727–1736.5. Stanyon, C. A., Liu, G., Mangiola, B. A., et al. (2004) A Drosophila proteininteractionmap centered on cell-cycle regulators. Genome Biol. 5, R96.6. Ito, T., Chiba, T., Ozawa, R., Yoshida, M., Hattori, M., and Sakaki, Y. (2001) Acomprehensive two-hybrid analysis to explore the yeast protein interactome.Proc. Natl. Acad. Sci. USA 98, 4569–4574.7. Schwikowski, B., Uetz, P., and Fields, S. (2000) A network of protein-proteininteractions in yeast. Nat. Biotechnol. 18, 1257–1261.8. Gavin, A. C., Bosche, M., Krause, R., et al. (2002) Functional organization ofthe yeast proteome by systematic analysis of protein complexes. Nature 415,141–147.9. Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., and Seraphin, B.(1999) A generic protein purification method for protein complex characterizationand proteome exploration. Nat. Biotechnol. 17, 1030–1032.