Editing Lentivirus Concentration by Ultracentrifugation (section)

= Protocol 1  =

Concentrate Lentivirus by ultracentrifugation.<br>Ultracentrifuge: Beckman LE 80K<br>Rotor: SW28<br>Tubes: #326823 autoclaved (polyallomer)

1. Pool all viral media into and filter through a 0.45ÎŒm HV Durapore membrane (Millipore #SCHVU01RE). <br>2. Place tubes into rotor holders and place on a scale. Slowly add ~37ml of virus/media to each tube and balance. (One 15cm plate should correspond to one ultracentrifuge tube).

Make sure the tubes are filled to about 1-3mm from the tube, or the tube will collapse while spinning.

3. Cap holders with a dab of grease and connect to the rotor.

4. Spin at 25,000 rpm (~82,000 Ã g) for 1 hour 45 min at 4°C. (It is not necessary to prechill the rotor.)

5. Carefully pour off supernatant and invert tube on kimwipes to dry (5-10â² at RT).<br>There should be a visible pellet.

6. Resuspend each pellet in 125ÎŒl of MEC growth media (or growth media for the cells you will be transducing).

Incubate at 4°C O/N to allow pellets to fully resuspend.

7. Combine all virus into 1 tube.

Dispense into 25ÎŒl aliquots and freeze at -80°C.<br><br>