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 .
