Amplification of Libraries

1. It is beneficial to screen unamplified libraries, since one needs to screen fewer clones; however, it is better to amplify the library for permanent storage. A good practice is to reserve a portion of the library unamplified and amplify the remainder for permanent storage.

2. It is convenient to plate 100-150 x 103 phage/fresh 150-mm plate. We prefer to plate in the morning and observe phage growth during the day. When plaques are touching, overlay each plate with 15 mL of SM, and incubate at 4°C overnight with shaking if possible. Plaques grown this way will typically be only 1-2 mm in diameter.

3. Harvest the liquid and make it 5.0% in chloroform. Be sure to use fresh chloroform that has been stored in the dark, because photodegradation products of chloroform are reportedly toxic to phage.

4. Spin out the debris, and transfer the lysate to an appropriate container. For storage at 4°C make the lysate 5% chloroform and store in the dark. A foil-covered Erlenmeyer flask, or media bottle is a good choice. The chloroform inhibits the growth of molds, which cause the library titer to drop rapidly.

5. It is probably a good idea to store aliquots of the library at -70°C for permanent storage. Bring the lysate to 7% DMSO, and freeze conveniently sized aliquots.

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