JoVE Encyclopedia of Experiments
Microbiology
0 views • 3:02 min • July 31st, 2026
Take an oncolytic virus-infected mammalian cell suspension.
Sonicate to lyse the cells and release the viruses into the solution.
Add DNase and its cofactor, then mix thoroughly and incubate.
The cofactor activates DNase, which degrades host DNA and reduces lysate viscosity.
Centrifuge the lysate to pellet host cell debris.
Collect the supernatant and filter it through a membrane to remove large host contaminants.
Repeat the filtration twice using membranes with progressively smaller pore sizes. This removes smaller contaminants while retaining the viruses in the filtrate.
Carefully layer the filtrate over a dense sucrose solution and centrifuge at high speed.
The viruses migrate to the bottom of the tube, while lighter contaminants remain in the supernatant.
Discard the supernatant and sucrose layer.
Add a stabilizing buffer that preserves viral integrity, then vortex to resuspend the viruses.
The purified viruses are now ready for further studies.
Thaw the previously snapped frozen cells in a warm water bath at 37 degrees Celsius and sonicate for one minute for three cycles in a water bath. Add 175 units of Benzonase Nuclease and two microliters of two millimolar magnesium chloride per milliliter of the cell lysate and vortex.
After an incubation of 30 minutes and warm water bath at 37 degrees Celsius, place the tube on ice. Spin the cell lysate at 300 times g for 10 minutes. Collect the supernatant in a new 50 milliliter conical centrifuge tube. Filter the supernatant through a sterile 5 micrometer PVDF membrane filter into a new 50 milliliter centrifuge tube called tube one. Add 1 milliliter of VB to the 50 milliliter centrifuge tube, emptied after filtering the supernatant from the previous step, vortex, and pass it through the same PVDF membrane filter placed on tube one.
Pass the filtrate from tube one through a sterile 0.8 micrometer MCE membrane filter placed on a new 50 milliliter centrifuge tube called tube two. Add 1 milliliter of VB to the emptied tube one, vortex, and pass it through the same MCE filter placed on tube two. Pass the filtrate from tube two through a sterile 0.45 micrometer PVDF filter placed on a new 50 milliliter centrifuge tube labeled tube three. As described before, repeat the washing of tube two with 1 milliliter of VB to add filtrate to tube three.