Purifying Viruses Using Sucrose Cushion
Prepare sucrose ( Molecular Biology Grade or higher ) as 38 % ( weight to volume ) in SM buffer ( 100mM NaCl , 8mM MgSO4 , 50mM TrisJHCl , pH 7.5 ) that has been 0.2μm filtered .
Rinse SW40 tubes with SM buffer or sterile water .
Place the 9.5ml DNase I - treated sample into the rinsed ultracentrifuge tube .
Using a 3cc syringe and cannula , slowly layer the 2.5 ml of 38 % sucrose under the sample trying not to cause any mixing of sample and sucrose .
Centrifuge at ~ 175,000g for 3.25 hr at 18°C .
Using a sterile transfer or serological pipet , pull off the 9.5 ml sample layer .
Pull off the interface and 2.5 ml sucrose , being careful not to disturb or suction off the pelleted sample at the bottom .
Keep this sucrose layer until virus counts from the pellets are completed .
Air dry the pellets in a fume hood for 15 - 20 min .
Add a mixture of TE containing 0.1M each EDTA / EGTA to resuspend the pellets ( 500 μl is recommended ) .
Perform virus counts and then extract DNA
