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328 Thibodeau-Beganny and JoungPCR conditionsCycling5 µL crude template 95°C for 5 min5 µL 10X Expand buffer 94°C for 30 s a(Roche)4 µL 10 mM dNTPs 60°C for 30 s a1 µL OK5 (10 pmol/µL) 72°C for 2.5 min a1 µL OK163 (10 pmol/µL) 72°C for 10 min0.375 µL Expand enzyme 33.625 µL H 2O(Roche)50 µLa Indicates repeated steps in 25 cycles.3. Run PCR products out on a 5% polyacrylamide gel or 1% agarose gel and isolatethe expected approx 1.8-kb DNA fragment. Sequencing of the target DNA-bindingsite can be performed using primer OK181.4. Typically, glycerol stocks and competent cells of the selection strain are alsoprepared using the overnight culture inoculated in step 1, Subheading 3.1.3.2.3.1.3.3. TRANSFORMATION OF RECOMBINANT F′ KJ1C STRAINWITH PLASMID PAC-αGAL4The final step in preparation of the selection strain is to transform the KJ1Cstrain, harboring a sequence verified F′ reporter episome, with the pAC-αGal4plasmid (13) (see Note 10).1. KJ1C cells bearing a sequence-verified recombinant F′ reporter episome aretransformed with plasmid pAC-αGal4 using standard chemical transformationprotocols. Transformations are plated on LB/CK plates because the recombinantF′ episome in the KJ1C cells confers resistance to kanamycin whereas the pACαGal4plasmid encodes a chloramphenicol resistance gene.2. Transformants are inoculated into overnight LB cultures containing chloramphenicol(30 µg/mL) and kanamycin (30 µg/mL) grown overnight at 37°C.3. Glycerol stocks of the final selection strains are prepared using the overnight culture.3.2. Selection of C2H2 ZFs Using the Bacterial Two-Hybrid SystemTo perform selections, phagemid libraries encoding randomized zinc fingersare introduced into a selection strain and then plated on selective media. Thesezinc finger variants are expressed as fusions to a fragment of the yeast Gal11Pprotein, which interacts with the fragment of the yeast Gal4 protein present inthe αGal4 protein expressed in selection strains. Binding of a zinc finger domainto the target DNA sequence leads to recruitment of RNA polymerase complexesthat have incorporated the αGal4 hybrid protein. This recruitment in turn leadsto activation of reporter gene expression and survival on selective medium.