Concentrated virus preps are often required for storage or downstream applications like infection and viral genome isolation. Precipitation methods have been developed to rapidly, easily, and cheaply concentrate viruses and remove impurities on the basis of solubility in solutions of inorganic salts or polyether compounds (e.g., ammonium sulfate, PEG-6000). The following protocol has been used for coronavirus enrichment via PEG precipitation.
Materials
Virus culture (preferably large-volume, and lacking serum and other protein supplements in the culture medium)
NaCl
PEG-6000
TES buffer:
NaCl 0.15 M
EDTA 0.002 M
Tris-HCl 0.01 M (pH 7.2)
Methods
1. Harvest the cells and culture medium by scraping with a rubber policeman.
2.Centrifuge at 3°C for 20 min:
10,000 rpm (15,300 × g) for a J2-21 centrifuge with a Beckman JA-14 rotor, or
12,000 rpm (15,000 × g) for a L8-70 centrifuge with a J-21 rotor
3. Transfer the supernatant to a large beaker in an ice bath atop a magnetic stirrer.
4. Slowly add NaCl (2.3 g of NaCl per 100 mL of supernatant) with constant, gentle stirring.
5. Slowly add PEG-6000 (7.0 g of PEG-6000 per 100 mL of supernatant) with constant, gentle stirring.
6. Cover the beaker and stir for 1 hour.
7. Incubate the beaker in the ice bath at 4°C overnight.
8. Repeat centrifugation as described in step 2.
9. Decant the supernatant, taking care not to disturb the precipitate.
10. Resuspend the precipitate in TES buffer (~2 mL per centrifuge bottle).
11. Aspirate thoroughly with a syringe and 22 G needle.
12. Transfer the suspension to a clean tube.
13. Rinse the bottle with TES buffer (~ 1 mL per bottle) and transfer to the tube containing the suspension.
14. Centrifuge at RT for 4 min (13,000 × g for a Beckman microfuge or similar instrument).
15. Save the supernatant (concentrated virus prep) and discard the pellet (PEG).
Reference
MAHY, B. W. J., & KANGRO, H. O. (1996). Virology methods manual. London, Academic Press.
**This is a suggested protocol and should be adjusted by the user accordingly**
While the above suggested protocol has been tested to be effective, if you are instead looking for a convenient solution for RNA isolation, complete with all buffers and reagents required, we recommend looking at the variety of viral & total RNA extraction kits that we have to offer:
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