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310 Serebriiskii et al.1. Use primers specific for the library plasmid (see Subheading 2.6.). Perform a PCRamplification (in ~50 µL volume with small amounts—barely visible—of the bacterialcolonies as PCR template), designing the amplification program as follows:a. 1 min—94°C.b. 45 s—94°C.c. 45 s—56°C (31 cycles of b–d).d. 45 s—72°C.In parallel with candidate colonies, set up PCR reactions from the followingcontrol templates: appropriate control plasmid (either pLibB-Raf or pTRG-RGLhighly diluted); E. coli carrying the same control plasmid (see Subheading 3.1.2.and Note 18).2. Run out an aliquot of the PCR reaction on a 0.7% agarose gel. Identify fragmentsthat appear to be of the same size. Digest some of the PCR-amplified DNA forthese clones with the frequently cutting restriction enzyme HaeIII, to determine ifan equivalent digestion pattern results: if so, the colonies are likely to be identical(see Note 19). Purified PCR fragments can be sequenced using the same primersused for amplification.3. For each potential-independent candidate, inoculate 2 mL of LB_A with bacteriapatched from a spot on the master plate created in Subheading 3.2.2., step 3.Grow at 37°C with agitation for 12 h or overnight.4. Isolate total plasmid DNA for each independent candidate interactor from 1.5 mL ofthe overnight culture using any standard miniprep isolation procedure. Resuspend orelute DNA in a final volume of 40 mL water. The plasmid DNA isolated by thismethod will include not only the prey plasmid, but also the bait plasmid as well.Hence, another round of transformation is necessary to separate the prey from the bait(see Note 20).5. Use 1 µL of the DNA from step 4 to transform a naive reporter strain (or anystandard E. coli cloning strain, such as DH-5α). To specifically rescue the libraryplasmid, spread 1/20th of the final transformation volume (or streak on a sectorof a plate using inoculation loop) on a LB_A plate in order to get single colonies.Incubate 16–18 h at 37°C.6. Pick two colonies from each transformation and using sterile toothpicks or tipspatch in an identical grid to an LB_C plate and an LB_A plate. Let patches grow6–8 h at 37°C.7. Transformants harboring only the prey plasmid should not grow on the chloramphenicol(LB_C) plate. For each candidate, pick one of the colonies that meet thiscriteria from the LB_A patch plate made in step 6 and inoculate a 10 mL cultureof LB supplemented with100 µg/mL ampicillin and grow at 37°C for 16–18 h withagitation.8. Isolate plasmid DNA from the 10 mL cultures using standard commercially availablealkaline lysis/column purification methods (or the means of one’s choice[17]). Utilize procedures for low-copy number plasmids, and perform all extrawash steps to obtain plasmid DNA of optimal yield and quality. This DNA can besequenced, or further confirmation tests (see Subheading 3.3.2.) performed.