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330 Thibodeau-Beganny and Joung16–18 h at 37°C and NM/CCK/50 µM IPTG plates for 24 h at 37°C. Titersfrom these plates can be calculated the next day after colonies have formed(see Step m).i. Pour some sterile glass beads (Fisher Scientific, cat. no. 11-312A) (3 mm) onto alarge (245 × 245 mm 2 ) NM/CCK/50 µM IPTG/10 mM 3-AT plate.j. Measure the remainder of the cell suspension, record the value (this volumewill be used when calculating titers), and then add the cells to the NM/CCK/50µM IPTG/10 mM 3-AT plate.k. Spread the cells with circular motions using the beads to distribute the cellsevenly.l. When plates have dried, turn plates over, and tap beads from the agar onto theinverted plate cover. Incubate at 37°C for 24 h and then at room temperaturefor 18 h.m. The next day, count colonies on the serial dilution plates to calculate the totalnumber of cells and total number of transformed cells plated on the selectionplate. LB/CK plates are used to determine the total number of cells platedand the LB/CCK and NM/CCK/I50 plates are used to determine the totalnumber of transformed cells plated. The following formula is used to performthese calculations:(No. of colonies/volume of spots [µL]) × dilution factor × volume (µL) platedon large dish.Example 65 total colonies in nine 5 µL spots of a 10 −6 dilution togetherwith 2650 µL plated on the large plate would give the following equation:(65 colonies/45 µL) × 10 6 × 2650 µL = 3.83 × 10 9 cells4. Recovery of zinc finger-encoding plasmids from cells surviving the selection. In thisstep, zinc finger-encoding plasmids from surviving cells are rescued as phagemidsby infecting these cells with M13K07 helper phage.a. Turn the large selection plate over and tap the glass beads back onto the agarand add 15 mL prewarmed NM media to the plate. Move the plates in a circularmotion using the glass beads to resuspend the cells in the media.b. Transfer the suspension to a sterile 25-mm glass tube.c. Remove 3 mL of cell resuspension to make glycerol stocks in case this recoverystep needs to be redone.d. Add enough of the cell suspension to 90 mL 2XYT supplemented with carbenicillin(50 µg/mL) and kanamycin (30 µg/mL) to give it a prelog appearance(i.e., an OD 600of ~0.1). Shake this culture at 120 rpm, 37°C for 1 h.e. Infect the log-phase culture with 10 12 kanamycin-transducing units of M13K07helper phage. Allow the phage to adsorb to the cells, without shaking, at roomtemperature for 30 min.f. Add kanamycin to a final concentration of 100 µg/mL (including the original30 µg/mL present in the culture). Shake the culture at 125 rpm, 37°C for 6 h.During this incubation, zinc finger-encoding phagemids from the cells will bepackaged as infectious phage particles harboring single-stranded DNA moleculesand extruded into the culture medium.