Cesium Chloride Purification of T7

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(Protocol)
(Protocol)
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#Add NaCl to the lysate to make the final concentration 1 molar.  Centrifuge the lysate at 10,000 rpm for 10 min, Discard the cellular debris, and add 10 grams polyethylene glycol (PEG) m.w. 8000 (10% w/v) to the supernatant.  Gently stir the mixture until the PEG has totally dissolved.  Keep lysate on ice for 1 hour.
#Add NaCl to the lysate to make the final concentration 1 molar.  Centrifuge the lysate at 10,000 rpm for 10 min, Discard the cellular debris, and add 10 grams polyethylene glycol (PEG) m.w. 8000 (10% w/v) to the supernatant.  Gently stir the mixture until the PEG has totally dissolved.  Keep lysate on ice for 1 hour.
#* Need to check the 1 hour incubation step because of warning from Studier on leaving cultures too long in the cell debris.  See [[Studier Lysate Prep]]
#* Need to check the 1 hour incubation step because of warning from Studier on leaving cultures too long in the cell debris.  See [[Studier Lysate Prep]]
-
#Pellet the phage at 5,000 rpm for 15 min.  Decant the supernatant, and very gently resuspend the pellet in 3.5 mL of [[T7 buffer]].  Centrifuge the lysate at 5,000 rpm for 10 min, and keep the supernatant.
+
#Pellet the phage at 5,000 rpm for 15 min.  Decant the supernatant, and very gently resuspend the pellet in 3.5 mL of [[T7 Buffer]].  Centrifuge the lysate at 5,000 rpm for 10 min, and keep the supernatant.
#Pour a cesium chloride step gradient: add 0.5 mL of cesium chloride with a density of 1.6 to the bottom of a centrifuge tube that fits in a SW 40.1 rotor.  Gently layer 0.5 mL of cesium chloride ρ=1.5 onto the ρ=1.6 layer.  Finally add 0.5 mL of cesium chloride ρ=1.4 onto the ρ=1.5 layer.
#Pour a cesium chloride step gradient: add 0.5 mL of cesium chloride with a density of 1.6 to the bottom of a centrifuge tube that fits in a SW 40.1 rotor.  Gently layer 0.5 mL of cesium chloride ρ=1.5 onto the ρ=1.6 layer.  Finally add 0.5 mL of cesium chloride ρ=1.4 onto the ρ=1.5 layer.
#Gently layer the phage supernatant onto the cesium chloride step gradient.  Centrifuge the phage in a SW 50.1 rotor at 30,000 rpm for 2 to 3 hours.  The phage will band at the ρ=1.5 layer.
#Gently layer the phage supernatant onto the cesium chloride step gradient.  Centrifuge the phage in a SW 50.1 rotor at 30,000 rpm for 2 to 3 hours.  The phage will band at the ρ=1.5 layer.

Revision as of 14:52, 3 June 2005

Summary

Protocol

  1. Grow 100mL of permissive cells to a density of 108 to 109 cells/ml at 30°C in a rotary shaking water bath. Inoculate the cells with a drop from a master phage stock. Continue to shake cells in the water bath at 30°C until culture clarifies. Be careful not to let culture sit for long (>15 minutes) after culture clarifies.
  2. Add NaCl to the lysate to make the final concentration 1 molar. Centrifuge the lysate at 10,000 rpm for 10 min, Discard the cellular debris, and add 10 grams polyethylene glycol (PEG) m.w. 8000 (10% w/v) to the supernatant. Gently stir the mixture until the PEG has totally dissolved. Keep lysate on ice for 1 hour.
    • Need to check the 1 hour incubation step because of warning from Studier on leaving cultures too long in the cell debris. See Studier Lysate Prep
  3. Pellet the phage at 5,000 rpm for 15 min. Decant the supernatant, and very gently resuspend the pellet in 3.5 mL of T7 Buffer. Centrifuge the lysate at 5,000 rpm for 10 min, and keep the supernatant.
  4. Pour a cesium chloride step gradient: add 0.5 mL of cesium chloride with a density of 1.6 to the bottom of a centrifuge tube that fits in a SW 40.1 rotor. Gently layer 0.5 mL of cesium chloride ρ=1.5 onto the ρ=1.6 layer. Finally add 0.5 mL of cesium chloride ρ=1.4 onto the ρ=1.5 layer.
  5. Gently layer the phage supernatant onto the cesium chloride step gradient. Centrifuge the phage in a SW 50.1 rotor at 30,000 rpm for 2 to 3 hours. The phage will band at the ρ=1.5 layer.
  6. Remove the phage band from the side of the tube with a syringe.
  7. Remove the cesium chloride by dialysis against 0.5 to 1 Liter of T7 Buffer at 4°C.

Reference

Luís René García

Ph.D. thesis with Ian Molineux

UT Austin

Characterization of Bacteriophage T7 DNA Entry into Escherichia coli

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