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Protocol

Preparation of M13 Single-stranded Template
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Isolation of phage DNA by Polyethylene Glycol (PEG) Transfer with plaques with a sterile toothpick into a 1-2 ml of 1X YT medium (RlO) in a sterile 1059 Falcon tube. To increase the yield of DNA add 10 ul of a fresh exponentially growing culture of JM103. Incubate at 370C with shaking for about 6-8 hours. Pour into a 1.5 mil microfuge tube and microfuge for 10minutes. Pour the supernatant into Another 1.5 ml tube, making no effort to transfer completely. The supernatant (0. 8) may be stored at 40C (not frozen) , but must be recentrifuged to remove any cells which grew during storage. The pellet is stored at 40C as a stock for that clone. To the culture supernatant (0.8 ml) add 200 ul 20% PEG, 2.5 M NaCl and incubate at room temperature 15 minutes. Microfuge for 5 minutes. Decant off the supernatant and wipe the inside walls of tube with Kimwipe. Add 100 ul TES buffer to pellet and suspend by vortexing for 2 seconds. Extract the suspended virus particles with 50 ul phenol (R2) followed by vortexing for 2 seconds. Let the extract stand for 5 minutes and vortex again. Microfuge for 4 minutes. With the aid of a pipetman set at 80 ul, remove 80 ul of the upper aqueous phase being very careful not to bring along any phenol or interphase. Add 3 ul 3M Na acetate (R4) and 200 ul ethanol. Place at -700C for 10 minutes or -200C overnight. Collect precipitated viral DNA by a 10 minute centrifugation in the microfuge. Pour off the supernatant and layer 1 ml cold ethanol on the pellet (do not mix). Recentrifuge 5 minutes, pour off ethanol and drain onto a Kimwipe. Dissolve the viral DNA in 50 ul TES (Rl8) (0.2-1 ug/ul). Store at -200C. lx TES: 20 mM Tris-HCl (pH 7.5), 10 mM NaCl, 0.1 mMNa2 EDTA Maxi Prep for Plasmid DNA Using Qiagen columns (based on 500 ml cultures) 1. Pour culture into 1 750 ml Beckman plastic bottles. 2. Spin for 351 at 3000 R.P.M. 40C. 3. Pour off supernatant. 4. Resuspend pellet in 10 ml of Buffer Pl. 5. Divide resuspension into 2 35 ml SW27 tubes. 6. Add 5.0 ml Buffer P2 to each tube. 7. Mix by inversion - gently. 8. Incubate at R.T. for 51. 9. Add 5.0 ml Buffer P3 to each tube. 10. Mix by inversion - gently. 11. Spin for 301 at 40C at 15,000 R.P.M. in SW27 tube (ultracentrifuge). 12. Combine supernatants. 13. Pre-equilibrate column with 5.0 ml of Buffer QB (2 drops/sec) . 14. Apply supernatant with 35cc syringe to column (1 drop/sec). 15. Wash 2x with 10.0 ml of Buffer QC (2 drops/sec). 16. Elute DNA with 5. 0 ml of Buf f er QF (1 drop/sec) into 15 ml tube. 17. Add 0.8 volumes of isopropanol. 18. Stand R.T.-201. 19. Spin at R.T. for 301 at 9500 R.P.M. in HB-4 rotor. 20. Wash pellet with 70% ethanol. 21. Dry pellet. 22. Resuspend in suitable volume of Buffer. Buffers 1. P1 2. P2 Standard Alkaline Lysis 3. P3 Pl + 400 ug/ml RNAse 4. QB 750 mM NaCl 50 mM Mops 15 % Ethanol pH 7.0 5. QC 1000 mM NaCl 50 mM Mops 15 % Ethanol pH 7.0 6. QF 1200 mM NaCl 50 mM Mops 15 % Ethanol pH 8.0 Storage of Buffers Buffer Pl at 40C. All others can be stored at R.T.
DNA Prep This is a modif ication of the standard Hirt procedure to purify viral or plasmid DNA from mammalian cells (JMB 26:365, 1967). DNA recovery is variable; recovery is poor for DNA molecules larger than 20-25 Kb. The DNA purified by this procedure is clean enough to digest with most restriction enzymes tested; but there is some material present that interferes with efficient transformation of E. coli with plasmids prepared in this way. Materials 1. PBS (or a similar physiological buffer). 2. Hirt lysis buffer: 0.6% SDS, 10 mM Tris, 10 mM EDTA pH 7.5-8. Keep at room temp. 3. 5 M NaCl. 4. RNase A: 2 mg/ml. 5. Phenol-Sevag: phenol 50%, chloroform 48%, isoamyl alcohol 2%. 6. phenol, ether and isopropyl alcohol. Methods: 1. Wash 9 cm confluent plates with lx PBS at room temperature. 2. Add 2 ml Hirt lysis buffer (1 ml if cells are not piled up). 3. Out of hood--sit at room temp. for 10-201, tilt plates occasionally to evenly distribute buffer. 4. Scrape lysate into 12 ml falcon (snaptop) tube. 5. Pour into SW50.1 polyallomar tube. 6. Add 1/4 volume 5 M NaCl. Cover top with parafilm. 7. Invert gently 6x. 8. Let it sit at least for 8 hours at 40C. 9. Spin (40C) at 18,000 rpm for 401 in precooled SW50.1 or SW55 rotor. 10. Transfer supernatant into 12 ml falcon tube. 11. Add 25 of RNase A. 12. 1 hour at 370C. 13. Phenol extract 1 x Phenol-Sevag extract 1 x Ether extract 1 x 14. Add NaOAc to 90 mM final concentration. 15. Add 5 ml isopropyl alcohol. Leave it overnight at -200C. 16. Spin 301 at 7.5K (,7A20 or a similar rotor). 17. Rinse pellet with cold 70% ETOH, repeat spin. 18. Vacuum dry. Dissolve in 100 sterile TE. 1 of this mateial from cells containing BPV-PBR plasmids gives 25-100 bacterial transformants (DH1). A much larger amount may inhibit transformation.
Preparation of M13 Single-stranded Template

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