Purification of viruses from culture lysates
Centrifuge the lysate at 4000g for 30 min .
Carefully decant and retain the supernatant .
Dissolve 8 % PEG ( w / v ) in clarified lysate and allow to precipitate overnight at 4°C .
Centrifuge the PEG solution at 10,000g for 20 min .
Carefully decant the supernatant , retaining the pellet .
Resuspend pellet in a small volume of residual PEG solution and pool all pelleted material .
Repeat steps 5 - 6 as needed to concentrate virus to < 1 mL .
Resuspend virus in 10 - 50 volumes of culture media to dilute PEG and allow virus pellet to disaggregate overnight at 4°C .
Concentrate sample to ~ 1 mL through a 30 kDa cutoff disposable centrifugal ultrafiltration device .
Prepare OptiPrep solutions using culture media as the diluent .
Using the underlayering technique with syringe and pipetting needle , pour 4 - step gradients into open - toped ultracentrifuge tubes , beginning with the least dense solution first .
Allow to blend for 2 h at room temperature .
Mark the top of the gradients with a fine - tipped marker , and carefully overlay virus concentrate using a transfer pipette .
Overlay culture media on balance gradients to create balance tubes .
Balance the tubes by adding media to underweight tubes .
Load tubes into rotor and ultracentrifuge at maximum permissible speed until density equilibrium is reached .
Using any fraction collection apparatus / technique , carefully extract purified viral concentrate from each tube .
If bands are not visible , a starting point is to fractionate the gradient into 4 + fractions , and use a couple of techniques to identify the virus - containing fraction ( i . e . , TEM , bioassay , absorbance at 260 nm , nucleic acid analysis , epifluorescence microscopy or flow cytometry ) .
