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282 Tikhmyanova et al.15. Prepare a master plate for each library plasmid being tested. Each plate should containat least 10 colonies of the transformed PCR-insert/digested plasmid into eachof a and b.16. Test for coloration and for auxotrophic requirements exactly as described forSubheading 3.3.5. above. True-positives should show an interaction phenotypewith a, but not with b. Yeast transformed with control PCR product will provideboth positive and negative controls: a should be negative whereas b should be positivewhen assayed for both color and growth on the corresponding plates (seeSubheading 3.2., step 4).17. Proceed with sequencing and biological characterization. Most often, PCR providesample source of DNA for all subsequent cloning. If needed, transform selected positivesinto E. coli by electroporation (20) using 1–2 µL of the β-glucuronidasetreatedfrozen yeast (step 3), and isolate plasmid DNA from Ap R colonies.3.3.6.2. PLASMID ISOLATION APPROACH: ISOLATION OF PLASMIDS, TRANSFERTO BACTERIAThis protocol provides an alternative option to the basic protocol in case PCRtechnology is not readily available for use, or in case of failure to obtain a specificPCR product using the library vector primers. This protocol is based onlysing the cells with glass beads after the β-glucuronidase treatment, followedby plasmid transformation in E. coli and plasmid isolation, and plasmid retransformationinto yeast. A number of kits for yeast minipreps are commerciallyavailable, for example, from Clontech (Clontech Laboratories Inc., www.clontech.com, Mountain View, CA) and others.1. Take 1–2 µL from the β-glucuronidase-treated yeast suspension (see Subheading3.3.6.1., step 3) and transform the DNA by electroporation (20) into any standardE. coli strain (e.g., DH5α) selecting a medium containing ampicillin, because onlybacteria that have taken up a library plasmid will grow.2. Select at least two bacterial clones for each yeast clone, and prepare a small quantityof plasmid DNA (12) from each bacterial clone.3. Follow Subheading 3.3.6.1. from step 11 to the end essentially as described,except transform with purified library plasmids, instead of mixture of PCR productand digested library vector.3.4. Follow-up for Library ScreeningFollowing completion of the aforementioned specificity tests, the next step isto leave work with the two-hybrid system, and proceed to biological characterizationof the interaction in the appropriate organism for the bait. Such characterizationwill be necessarily bait specific, and should serve to further eliminateinteractions of dubious physiological relevance. Of note, a database of commonfalse-positives, along with discussion of issues related to false-positives, is foundat: http://www.fccc.edu/research/labs/golemis/interactiontrapinwork.html.