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Engineering Cys2His2 Zinc Finger Domains 329Typically, selections are performed in two stages. In stage A selections,large numbers of transformants (typically ~10 9 ) are plated on a low-stringencyselection plate. Zinc finger-encoding phagemids are rescued from survivingcells. In stage B selections, this enriched population of phagemids are thenreintroduced into the selection strain cells and plated on a series of higherstringency selection plates. Phagemid DNA is then isolated from cells thatgrow on the highest stringency plate and sequenced to determine the identityof the fingers.3.2.1. Stage A Selections1. Streak out the selection strain on an LB/CK plate and incubate overnight at37°C.2. Use a fresh, well-isolated colony to start an overnight culture of the strain in20 mL of NM media supplemented with chloramphenicol (30 µg/mL), kanamycin(30 µg/mL), and 50 mM IPTG. Because selection strain cells are sensitive to detergentsand rapid agitation, this culture should be grown in a sterile 125-mL glassflask that was rinsed thoroughly with deionized distilled water before autoclavingand with shaking at 110 rpm at 37°C for approx 16–24 h.3. Introduction of zinc finger phagemid libraries into selection strain cells. The constructionof the randomized zinc finger phagemid libraries used in this step hasbeen previously described (12,13).a. Thaw phagemid phage library on ice. Extreme care is required to prevent phagecontamination in the lab as it may persist.b. Transfer 5 mL of the saturated overnight culture of selection strain cells to asterile 125-mL flask.c. Add approx 6 × 10 8 ampicillin transducing units (see Note 11) of phagemidlibrary to the selection strain cells and gently swirl immediately. Allow thecell/phagemid mixture to sit at room temperature for 30 min without agitation.d. Add 20 mL of prewarmed NM media supplemented with chloramphenicol(30 µg/mL), kanamycin (30 µg/mL), and 50 mM IPTG to the infected cells.Shake at 110 rpm, 37°C for 90 min.e. Transfer culture to a sterile 50-mL conical tube and pellet cells by spinning at2500 rpm in a table top centrifuge for 25 min at room temperature.f. Pour off medium and resuspend the cell pellet in 2.5 mL of prewarmed NMmedium supplemented with chloramphenicol (30 µg/mL), kanamycin(30 µg/mL), and 50 mM IPTG.g. Serially dilute 100-µL aliquots of the cell resuspension in a sterile 96-wellmicrotiter plate (Corning, Cat. no. 3596). Perform three independent dilutionsets using NM medium supplemented with chloramphenicol (30 µg/mL),kanamycin (30 µg/mL), and 50 mM IPTG. Perform dilutions from 10 −1 to 10 −8(note that one will only plate dilutions 10 −3 –10 −8 ).h. Spot 5 µL of the 10 −3 to 10 −8 dilutions each in triplicate on LB/CK, LB/CCK,and NM/CCK/50 µM IPTG plates. Incubate LB/CK and LB/CCK plates for