Chlorovirus Purification
Inoculate flasks with NC64A chlorella in MBBM (or Pbi in FES, SAG 241-80 in MBBM) and incubate for several days at 25oC with continuous light and shaking.
Infect the flasks of chlorella with virus at an moi of 0.01 to 0.001.
Incubate the flasks for 48-72 hours at 25°C with continuous light and shaking.
Add Triton X-100 to the lysate supernatants to a final concentration of 1%.
This solubilizes the green pigment in the supernatant.
Stir this solution at room temperature for at least one hour.
Centrifuge the lysate in the Sorvall GSA rotor at 5,000 rpm (4,000 rcf), 5 min, 4°C.
Discard the pellets.
Centrifuge the lysate in the Beckman Type 19 ultracentrifuge rotor at 17,000 rpm (43,000 rcf), 50 min, at 4°C.
Discard the supernatants.
Resuspend the virus pellets with a small volume of 50 mM Tris-HCl, pH 7.8.
Adjust the resuspended virus material with Protease K to 0.02 mg/mL and incubate at 45oC for at least one hours.
For NC64A and Pbi virus lysates: Layer the virus suspension onto 100-400 mg/mL (10-40%, w/v) linear sucrose density gradients equilibrated with 50 mM Tris-HCl, pH 7.8, made up in Beckman SW28 rotor tubes.
For SAG 3.83 virus lysates: Layer the virus suspension onto ~100-400 mg/mL linear iodixanol gradients equilibrated with 50 mM Tris-HCl, pH 7.8, made up in Beckman SW28 rotor tubes.
Centrifuge the gradients in a Beckman SW28 or SW32 rotor at 20,000 rpm (72,000 rcfmax), 20 min, 4°C.
Remove the virus bands from the gradients with sterile bent needles via top (or via side puncture with sterile needle and syringe) to oak ridge 30 mL polypropylene tubes.
Split the virus from 3 gradients between 2 tubes.
Slowly dilute the virus to the tube volume with 50 mM Tris-HCl, pH 7.8.
Centrifuge the tubes in Beckman Ti 50.2 rotor at 27,000 rpm (~44,000 rcf), 3 hours, 4°C.
Discard the supernatants.
Resuspend the virus pellets with a small volume of 50 mM Tris-HCl, pH 7.8.
Store the virus at 4°C.
Do not freeze.
Layer the virus suspension onto 10-40%, w/v linear iodixanol or sucrose density gradients equilibrated with 50mM Tris-HCl, pH 7.8 made up in Beckman SW28 rotor tubes.
Centrifuge the gradients in Beckman SW28 rotors at 20,000 rpm, 4 hours, 25oC.
Remove the virus bands from the gradients with sterile bent needles via top (or via side puncture with sterile needle and syringe) to oak ridge 30 mL polypropylene tubes.
Split the virus from 3 gradients between 2 tubes.
Slowly dilute the virus to the tube volume with 50 mM Tris-HCl, pH 7.8.
Centrifuge the tubes in Beckman Ti 50.2 rotor at 27,000 rpm (~44,000 rcf), 3 hours, 4°C.
Discard the supernatants.
