protocol_166.txt

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