Packaging and Titering the Library

1. Package the ligation mix as directed in the instructions accompanying the packaging mix (Note13).

2. Dilute the packaged phage with 500 ||L of SM, and add 20 ||L of chloroform (not chloroform:isoamyl alcohol). Store in the dark at 4°C.

3. Plate 1, 10, and 100 |L of a 10-3 dilution on 300 |L of ER-1647 plating cells grown as described below. Adsorb the phage to the plating cells for 15-30 min at room temperature, and then transfer to 37°C for 5 min. Phage can adsorb to the receptors, but cannot inject their DNA at room temperature. The transfer to 37°C produces a relatively synchronous infection. Incubate at 37°C for 8 h to overnight. It is essential that the strain used be deficient in methylcytosine restriction (mcrA-, mcrBC).

4. Determining the fraction of recombinant clones in the library. Since ER-1647 is lac-, the library must be plated on an appropriate strain that allows a complementation and is McrABC-, like YS-1, or amplified first on ER-1647 and then plated on XL 1-Blue. After amplification, the fraction of recombinants can be determined by plating on XL1-Blue with X-gal and IPTG as described above. Using UniZap-XR, a typical yield is 95+% recombinant phages.

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