• NZCYM medium
  10g NZ Amine
   5g NaCl 
   1g Casamino acid  
   5g Bacto- yeast extract
   2g MgSO₄ ^. 7H₂O
   To 1 liter
• NaCl
• PEG  (polyethylene glycol)
• SM
  0.1 M NaCl
  0.05 M TRIS
  0.01% gelatin
  0.01 M MgSO₄
• CsCl
• 0.1 M TRIS pH 8.0
• 5M NaCl
• 1 M MgSO₄
• Sevag
  Chloroform :  Isoamyl alcohol (24:1)

Method

Phage Culture

1. Mix 1 x 10¹⁰ E. coli (e.g., K803 - fresh overnight culture best but not mandatory) with 5 x 10⁷ to 5 x 10⁸ pfu.
2. Preadsorb bacteria and phage 15 minutes at 37°C.
3. Add into 500 ml NZCYM medium.
4. Shake 250 rpm 4-8 hours until cell lysis is maximum.
5. Spin out debris for 10 minutes at 5,000 RPM in Sorvall GSA rotor.
6. Add 29.2 gm NaCl (1.0 M) to supernate.
7. Add 50 gm PEG(10% final w/v); Mix gently 15-20 minutes to dissolve; keep on ice whenever possible.
8. Keep > 2 hrs 4°C to precipitate.
9. Spin 5 minutes at 5000 RPM in Sorvall GSA rotor.
10. Drain pellet, wipe inside of bottle; work quickly so pellet does not dry out (and become difficult to resuspend).
11. Resuspend in approximately 4 ml SM/250 ml original culture medium.
12. Add 0.45 gm CsCl for each 0.55 ml SM + phage.
13. Mix gently until CsCl is dissolved.
14. Spin 24 hours in Sw 55 Ti, at 39K RPM, at 4°C.
15. Remove blue phage band by puncturing tube with needle and syringe.
16. Dialyze CsCl-purified phage in 200 ml 0.1 M TRIS pH 8.0, 50 mM NaCl, and 1mM Mg⁺⁺, 2 hours-overnight.

1. To the dialyzed phage, add EDTA pH 7.6.0 to 50 mM; SDS to 0.05%, and proteinase K to 100 µg/ml. Incubate 30 minutes at room temperatue.
2. Extract gently with phenol, phenol/sevag, and sevag.
3. Dialyze vs. 10 mM TRIS pH 8.0, 0.1 mM EDTA.