7.2.3.3. Fractionation

1. After centrifugation, carefully remove the centrifuge tubes from each bucket.
If there is a lot of virus then the virus particles can be seen as an iridescent band underneath a top-light (Figure 3).  
Often two bands can be seen; a lighter band higher in the gradient and a more intense band lower in the gradient. These correspond to ‘empty’ and ‘filled’ (with RNA) particles respectively.   

2. Remove the band(s) with a disposable syringe and needle.
This is best done using a needle with a ‘flat’ end, rather than the ‘angled’ end. Slide the needle along the centrifuge wall to just below the band and draw up the band slowly into the syringe.

3. If no bands can be seen, either because of low virus amounts or because the centrifuge tube is opaque, then the gradient needs to be fractionated by removing 0.5 ml volumes at a time.  
The best way is to use an automated fractionator, which removes the fractions from the bottom of the gradient. The manual alternative is to remove 0.5 ml fractions from the top of the gradient.

4. Analyse 10 µl of each fraction for the presence of virus, using either ELISA (see section 9.2.) or RT-(q)PCR (see section 9.3. and the BEEBOOK article on molecular methods; Evans et al., 2013).  
Since there will be some virus contaminating every fraction, qualitative RT-PCR will not be able to distinguish very well between high and low virus fractions. 

5. Pool the 3-4 fractions containing the highest virus concentrations, giving a final volume of approximately 2 ml. 

6. If necessary, the virus can be concentrated further by another high-speed centrifugation (Table 4), although this will reduce the yield.

Fig. 3. White translucent band containing DWV particles after CsCl density gradient centrifugation. Image © E Ryabov.

12116PN revised Fig 3