Library Screening

1. Plate the library to be screened at relatively low density (~5-10,000 phage/150-mm plate) to minimize the number of purification steps required. Concerning the amplification step described above, it is best to plate the phage in the morning, observe their growth during the day, and remove the plates to 4°C when plaques are relatively large (~2-mm), but still well isolated from each other.

2. Lift dupicate filters from each plate, the first for 3 min and the second for 6 min (see Note 19). Store the filters plaque side up on Whatman 3MM paper until all lifts are completed. Be sure to position registration marks carefully for accuracy in aligning the autoradiograms later.

3. Denature the phage by placing the filters (plaque side up) on pads of Whatman 3MM paper saturated with the following solutions for 3' each (10): Solution 1—0.5 N NaOH, 1.5 M NaCl; Solution—2 1.0 M Tris-HCl, pH 7.5, 1.5 M NaCl; Solution 3—2X SSC. Next, transfer the filters to 3MM paper, and allow to air-dry.

4. Interleave the filters between circles of filter paper, and secure the entire stack with tape. Bake at 80°C for 30 min.

5. After baking, transfer the filters to a dish of 50 mM NaOH, and incubate for 15 min with shaking. This step reduces later background. Rinse with at least four changes of distilled H2O for a total of 15 min.

6. Transfer the filters to a hybridization bag, and add 2 mL/filter of Church's buffer (7% SDS, 0.5 M NaPO4, pH 7.2) (16). Prehybridize at 65°C for a convenient time, usually 15 min to several hours. Up to 20 filters (132 mm) can be processed/bag.

7. Remove the prehybridization solution and add fresh Church's buffer containing 5% w/v Dextran sulfate (Pharmacia) at 750 pL/filter. Denature the probe by adding 0.1 vol 2 N NaOH and incubating at room temperature for 5 min. Add the denatured probe directly to the bag, seal, and mix well. Hybridize at 65°C overnight with shaking if possible.

8. Remove and save the hybridization solution at -20°C (add fresh probe to it for subsequent screening). Wash the filters 3 x 20' at 65°C in 0.5X SSC, 0.1% SDS. Expose to X-ray film with two intensifying screens overnight or longer.

9. Align the duplicate autoradiographs, and pick plaques with the wide end of a Pasteur pipet, which appear on both filters to 1 mL of SM buffer.

10. Plate several dilutions of each plaque stock, and incubate overnight at 37°C. Select a plate with ~50 plaques for the next round of screening. After this round, the individual plaques should be pure and can be picked with the narrow end of a Pasteur pipet.

11. Convert the phage to plasmids according to the Stratagene protocol provided with the ZAPII vector or library.

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