Chlorella NC64A and Mictractinium Pbi virus Purification
Inoculate flasks with Chlorella NC64A in MBBM (or Micractinium Pbi in FES) and incubate at 25°C with continuous light and shaking until the cells are in the actively growing phase (about 1-2 X 107 cells/ml).
Infect the flasks of chlorella with virus at a multiplicity of infection (moi) of 0.01 to 0.001.
Incubate the flasks for 48-72 hours at 25°C with continuous light and shaking.
This material is now termed “lysate”.
Centrifuge the lysate in the Sorvall GSA rotor in 250 ml bottles at 5,000 rpm (4,000 rcf), 5 min, 4°C.
Discard the pellets.
Add Triton X-100 to the lysate supernatants for a final concentration of 1% (from a 10 or 20% stock).
Centrifuge the lysate in the Beckman Type 19 225 ml 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 (approximately 1.0 mL per 100 mL of original lysate).
Layer the virus suspension onto 100-400 mg/mL (10-40%) linear sucrose density gradients equilibrated with 50 mM Tris-HCl, pH 7.8, made up in Beckman SW28 rotor tubes (layer approximately 3-4 mL per gradient).
Centrifuge the gradients in a Beckman SW28 rotor at 20,000 rpm (72,000 rcfmax), 20 min, 4°C.
Remove the virus bands from the gradients with sterile bent needles and transfer to oak ridge 30 mL polypropylene centrifuge 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.
Gently wash the pellet and bottle with some 50 mM Tris, pH 7.8 buffer to wash residual sucrose away.
Store the virus at 4°C.
Do not freeze.
Filter sterilization using a 0.45 µm cellulose acetate or other low protein binding filter is recommended.
Then resuspend the virus pellets with a small volume of 50 mM Tris-HCl, pH 7.8.
