RAPID PURIFICATION OF PHAGE USING THE TL-100

RAPID PURIFICATION OF PHAGE USING THE TL-100

  1. Place approximately 106 phage particles on a 23x23 cm agar plate which has been seeded with \-receptive E. coli.
  2. Incubate 6 hr at 37C or 3-4 hr at 42C, depending on the phage.
  3. Collect the top agar and clarify by centrifugation at 500-800X g for 5 min.
  4. Remove the supernatant (approximately 2 ml) and extract twice with CHCl3. Then, if the volume of supernatant was larger than 2 ml, precipitate with 1 M NaCl-10% PEG 6000 for 30 min on ice. Resuspend in 2.0 ml of Tris-MgCl2 buffer (1 mM Tris (pH 7.5), 1 mM MgCl2).
  5. Add 1.42 g CsCl for every 2 ml of supernatant and dissolve thoroughly. The density of the final solution should be 1.46 g/ml with respect to CsCl.
  6. Centrifuge using the TLS-55 at 55,000 rpm for 2 hr. The phage will band in the middle of the tube between the bacterial DNA and the RNA pellet.
  7. Extract the DNA using an appropriate method, and dialyze against Tris-EDTA buffer.