Phage Minipreps

1. In order to prepare synthetic RNA from a XZAPII library, one must first prepare a sufficient quantity of phage DNA. With current RNA preparation technology, ~10 |g should be sufficient.

2. A high titer library (1010 PFU/mL) will yield about 500 ng of phage DNA/mL, so 20-50 mL is about the right amount.

3. Add DNase I and RNase A to 50 |g/mL. Incubate at 37°C for 1 h with gentle shaking.

4. Transfer to a centrifuge tube, and spin at 12,000g for 10 min. Carefully decant the supernatant to a fresh tube.

5. Add 1/4 vol of 20% PEG 8000, 2.5 M NaCl, mix well, and incubate on ice for 1 h. Spin at 12,000g for 10 min at 4°C. Carefully remove the supernatant taking care not to dislodge the phage pellet.

6. Resuspend the phage in a minimum volume of proteinase K reaction buffer (10 mM Tris, pH 8.0, 5 mM EDTA, 0.5% SDS). Take care to resuspend well at this point for maximum yields.

7. Transfer to a 1.5-mL microfuge tube, and add proteinase K to a final concentration of at least 200 |g/mL, and preferably 1 mg/mL. Incubate at 60°C for >30 min or 37°C overnight (see Note 15). Be sure that the phage do not clump up during the digestion. Pipet up and down if they do, or you will lose them in the phenol extraction.

8. Phenol:chloroform:isoamyl alcohol-extract and back-extract the organic phase with 20% of the original volume of TE. Pool the two aqueous phases.

9. Repeat the phenol extraction.

10. Transfer the aqueous phase to a fresh tube, and add 2 vol of EtOH. Now add 0.5 of the original volume of 7.5 M NH4-acetate and mix well (see Note 16). Mix well and store on ice for 30 min. Spin for 20 min at 4°C. Rinse the pellet well and dry.

11. Resuspend the pellet in a minimum volume of TE, quantitate, and check a small aliquot for purity by agarose gel electrophoresis.

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