Propagation of marine eukaryotic viruses ( Prasinoviruses )
Cleaning and Concentration of Lysate .
Once the primary infection culture has cleared , filter entire volume of lysate through a 0.45 - µm - pore - size PES membrane sterile disposable filter unit to remove large cell debris .
Add 20 mL filtered lysate to the upper reservoir of a 100,000 Dalton MWCO PES membrane Vivaspin20 ultrafiltration unit .
NOTE : Appropriate MWCO will depend on virus capsid size .
For maximum recovery select a MWCO at least 50 % smaller than the molecular size of the particle of interest .
200 kDa is the equivalent of ~ 10 nm .
Place unit with counter - balance in swinging bucket rotor in pre - cooled ( 4°C ) benchtop centrifuge so that the printed side faces upwards / outwards .
Centrifuge at 1,000 x g ( do not exceed ! ) for 5 - 10 minutes .
Centrifuge time will depend on the amount of material in the sample .
Do not let the filter go dry .
Record the volume retained in the upper reservoir .
Discard the filtrate by separating the upper reservoir from the bottom of the Vivaspin20 unit .
Repeat 2 - 6 ( adding filtered lysate to the retentate in the upper reservoir up to 20 mL before re - centrifuging ) until the entire volume of filtered lysate has been processed .
NOTE : Several Vivaspin20 units can be used in parallel for large lysate volumes , and pooled after concentration .
Transfer the concentrated virus sample ( retentate ) from the upper reservoir to a sterile 15 or 50 mL conical tube .
Remove the upper reservoir from the Vivaspin20 unit and cover the bottom with a double - layer of Parafilm .
Add 2 mL of 0.02 - µm - filtered 1X TE buffer ( pH 8.0 ) to the upper reservoir .
Vortex the upper reservoir ( Parafilm side down ) for ~ 20 sec on 60 % power to wash the Vivaspin20 filter .
Add the washed sample to the recovered viral concentrate .
Repeat 10 - 12 twice more for a total of 3 washes with TE buffer .
Store concentrated virus sample at 4°C protected from light .
Primary Infection .
Ethanol - clean culture hood as usual , then UV pipettes , tips , and tube rack for 10 min .
Work with one virus at a time , transferring the master stock from 4°C to the culture hood .
Initiate the primary infection by adding the master virus stock at 1 % of the total culture volume ( e . g . , 5 mL virus to 500 mL exponentially - growing host culture ) .
Mix the infected flask and incubate at standard growth conditions until culture is mostly lysed ( e . g . , ~ 5 days for O . lucimarinus viruses ) .
Thoroughly clean pipette and gloves with ethanol before repeating for additional viruses .
Ethanol - clean hood and UV pipettes , tips , and tube rack after use .
