Viruses Purification of Perkinsus spp .
First , Fill centrifuge bottles with 450mL Perkinsus Culture in the logarithmic growth phase .
Centrifuge bottles at 1500 g ; 15 min at room temperature .
Transfer supernatant to a clean tube , and store at 4°C for further analyses .
Resuspend pellet in 10 % initial culture volume supernatant in a 50mL Falcon Tube .
Centrifuge tubes at 1500 g ; 15 min at room temperature .
Transfer supernatant to a clean tube , and store at 4°C for further analyses .
Resuspend pellet in 4mL PBS / 0.35 M NaCl ) containing protease inhibitor cocktail .
Transfer the pellet resuspended in 2mL Eppendorf Tubes ( 1.5mL / tube ) .
Add 100mL Glass beads .
Disruptor Genie for 15sec .
Verify cell disruption : place a 20mL under light microscope .
Disruptor Genie for 1min .
Verify cell disruption : place a 20mL under light microscope .
Disruptor Genie for 3min .
Verify cell disruption : place a 20mL under light microscope .
Centrifugation at 10,000 g ; 30 min , 4°C .
Removed the supernatant to clean 1.5mL Eppendorf tube , discard the pellet ( cell debris ) .
Centrifugation again at 10,000 g ; 30 min , 4°C .
Removed the supernatant to clean 1.5mL Eppendorf tube , discard the pellet ( cell debris ) .
Conserve the tubes at 4°C
