ReadiPrep™ PEG Virus Precipitation Kit

The procedure is designed for 10 mL of virus solution, but one can adjust the volumes proportionally based on the sample size.

1. Infect cells or transfect cells, and allow maximum virus accumulation.

2. For mammalian cell-virus or insect-baculovirus, centrifuge culture at 3,200 X g (for bacterial phage, centrifuge at 16,000 X g) for 15 minutes at 4 °C to remove cell debris.

3. Collect supernatant, and add 2.5 mL of ReadiPrep™ 5X PEG Solution (Component A) to 10 mL of virus supernatant.

4. Refrigerate overnight (stable up to 2 days at 4 °C).

5. Centrifuge at 3,200 X g for 30 minutes at 4 °C.

6. Remove and discard the supernatant by aspiration carefully. The beige or white pellet is the virus.

7. Add 100 uL of ReadiPrep™ Virus Resuspension Solution (Component B) to the virus pellet, and mix well.

8. Aliquot the virus and store it at < –70 °C for future use.

   Note: For a high-titer virus preparation, the resuspension volume should be limited to about three times the volume of the white pellet, usually 1/10 to 1/100 the volume of the original sample. If insoluble material is present in the viral suspension, it can be removed by centrifuge at 3,200 x g for 15 min at +4 °C.

   Note: Avoid freeze/thaw cycles to maximize virus recovery.

   Note: Trace amounts of PEG in the virus suspension will not affect the use of the concentrated virus. In some cases, PEG may increase virus infection efficiency. However, if it is desired, the trace amount of PEG can be removed by the following procedure:

   1. Add 1 volume of solution containing 4 M KCl and 50 mM Tris-HCl, pH 7.2 (not provided), to 3 volumes of the concentrated virus suspension.  Alternatively, add solid KCl into the virus suspension to a final concentration of 1 M.

   2. Let stand on ice for 15–30 minutes. Then, spin at 12,000 x g for 10 min at 4 °C to remove the precipitate.

   3. Collect the virus supernatant carefully. And aliquot and store at < –70° C for future use.