126.96.36.199. Sucrose gradients
1. Prepare four solutions,
containing the appropriate virus extraction buffer (Table 4) and either 10%,
20%, 30% or 40% sucrose.
This is best done by mixing the appropriate amounts of 10x buffer, 60% sucrose solution and water.
2. Divide the total volume of a centrifuge tube by 5.
3. Add 1/5th volume of the 10% sucrose-buffer solution to every centrifugation tube.
Be accurate with the volumes, to avoid problems with balancing the tubes later-on.
4. Using a syringe with a long needle, layer 1/5th volume of the 20% sucrose-buffer solution underneath the 10% solution.
5. Repeat with 1/5th volumes of the 30% and 40% sucrose-buffer solutions.
6. Place the tubes in a freezer with minimum disturbance,.
7. Once completely frozen, take the tubes out and thaw completely.
The higher concentration solution will thaw earlier than the lower density solutions, causing the boundaries between the concentration layers to blur.
8. Repeat the freeze-thaw process twice more to extend this process, generating a continuous density gradient.
Do not freeze-thaw too often, or you will end up with a single-density solution.
9. For discontinuous gradients, layer 1/5th volume of the 40% sucrose-buffer solution underneath 3/5th volume of the 10% sucrose-buffer solution.
10. Layer 1/5th volume of virus extract carefully on top of the gradient.
11. Balance the tubes by weight to within 1 mg, using buffer solution.
12. Insert tubes carefully in the buckets and hang the buckets in the correct orientation in their appropriate place on the rotor.
13. Centrifuge in a swing-out rotor at the appropriate speed, time and temperature for the virus in question (see Table 4).