Ultracentrifuge: Beckman LE 80K
Tubes: #326823 autoclaved (polyallomer)
1. Pool all viral media into and filter through a 0.45μm HV Durapore membrane (Millipore #SCHVU01RE).
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 × 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 (5-10′ at RT).
There should be a visible pellet.
6. Resuspend each pellet in 125μl 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 25μl aliquots and freeze at -80°C.