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 .
