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Introduction:
Virus was usually produced at a low titer. It often needs to be concentrated for storage or further applications. A quick, easy and inexpensive method is desired to concentrate virus and remove impurities. BioVision’s PEG Virus Precipitation Kit provides an easy, convenient and time-saving method to concentrate virus without ultra-centrifugation. The kit can be used for small lab samples or large scale virus preparation with high yield and high viral titer. The kit can be used to concentrate retroviruses, baculoviruses, lentiviruses, and phages etc. in cell culture medium or environmental samples. Virus can be concentrated over 100 folds. An optimized Virus Re-suspension Solution is provided to maximize viral
recovery by 40-100% depending on the virus type and sources. The whole process uses non-toxic reagents. The concentrated virus can be used for infection, viral DNA or RNA purification, etc.
Reagent Storage Conditions:
The solutions are ready to use and stable for 12 months at +4°C or at -20°C for long term storage.
Protocol:
Step-I：Virus production
1 Infect cells and allow to incubate for as long as necessary to approach maximum virus production for the strain and cell type combination.
2, Centrifuge culture at 1000rpm for 7 minutes.
3, Resuspend cell pellet in fresh media. Incubate for an additional 48hrs.
4, Centrifuge culture at 14000rpm for 15 minutes to remove cells.
5, Collect supernatant and add 1 volume of PEG solution to 4 volumes of virus containing supernatant.
6, Refrigerate overnight (stable up to 2 weeks at 4°C ).
7, Centrifuge at 2300rpm for 45 minutes.
8, Remove supernatant.
9, Resuspend virus pellet in 1/10 to 1/100 of original volume.
10, Aliquot in small aliquots and store at -70°C until use.
Notes:
A). For high titer virus preparation, the re-suspension volume should be limited to about three times the volume of the white pellet, usually 1/10 to 1/100 volume of original sample. If insoluble material is present in the viral suspension, it can be removed by centrifuge at 3,200xg for 15 min at 4oC.
B). Avoid freeze and thaw cycle to maximize virus recovery.
C). Trace amount 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 following procedure: Add 1 volume of solution containing 4 M KCl and 50 mM Tris-HCl, pH7.2 (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. Sit on ice for 15-30 min. Spin at 12,000xg for 10 min at 4°C to remove the precipitate. Carefully collect the virus supernatant. Aliquot and store at -70°C for future use.
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