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Screening in Yeast With a Bacterial/Yeast Two-Hybrid System 281fragments that appear to be of the same size; HaeIII digests of these fragmentsshould be run side-by-side. Put gel in a refrigerator until it is ready to isolate fragments(see Note 29).6. Perform a restriction digest of 10 µL of the PCR product with HaeIII in a total volumeof 20 µL. Rearrange the loading order according to the results obtained with nondigestedPCR, and load the digestion products on a 1.5% agarose gel. Run out the DNAa sufficient distance to get good resolution of DNA products in the 200–1000 bp sizerange. This will generally yield distinctive and unambiguous groups of inserts, confirmingwhether multiple isolates of a small number of cDNAs have been obtained.7. Purify the uncut fragments from the gel by using standard agarose gel-purificationtechniques. In cases whereby a very large number of isolates representing asmall number of cDNA classes have been obtained, the investigator mightchoose to directly sequence the PCR product (see Note 30). The purified cDNAcan be used directly to reassess the interaction with bait (second confirmation ofinteraction).8. The next step is to establish whether isolated cDNAs are reproducible and able toreassess, whether interactors associate specifically with the bait(s) of interest, orare nonspecific (“sticky” proteins), or false-positives that were spuriously isolatedowing to mutations in the initial bait strain that lead to nonspecific growth and/ornonspecific transcriptional activation. This can be done using a PCR-recombinationapproach (derived from ref. 25) in a single step, after which confirmed specificpositive clones can be worked up through conventional plasmid purification.9. Perform a restriction digestion of an empty library plasmid with two enzymes producingincompatible ends in the polylinker region (e.g., EcoRI and XhoI), see Note 31.10. Perform PCR from positive control plasmid(s) using the same primers as before,and purify the PCR product.11. Transform PRT50 containing pGLS22-Ras + pLacGus + pEG202-Krev1 (seeNote 19) with:a. Digested library plasmid (50–100 ng).b. Digested library plasmid (50–100 ng) and control PCR product (0.5–1 µg).c. Uncut library plasmid (50–100 ng).Save the extra digested library plasmid and the pPrey-control PCR product forfurther use in the specificity test (step 13).12. Plate the transformations on Glu/CM Ura − His − Trp − dropout plates and grow at30°C for 2 d (until colonies grow). Count colonies (see Notes 32 and 33). If transformationefficiency in b is better than in a by 5–20-fold, it is safe to proceed tothe next steps. c is a positive control for the transformation (Note 34).13. Using same ratios as in step 11b above, transform digested library vector in combinationwith selected PCR products (again, include positive control(s) from step 9)to the following:a. pGLS23-Bait1 + pLacGus + pMW103-Bait2 (the original naïve bait strain).b. pGLS22-Ras + pLacGus + pEG202-Krev1 (the control bait strain).14. Plate each transformation mix on Glu/CM Ura − His − Trp − dropout plates and incubateat 30°C until colonies grow (2–3 d).