Experimental Reagent Extraction Buffer I (GMT) [Details:
Glycine 0.3mol / l
MgCl2 0.03mol / l
Tris-HCl 0.05mol / l (pH 7.5)]
Extraction Buffer II (PB) [Details:
Na2HPO4 0.1mol / l
KH2PO4 0.1mol / l (pH 5.8)
PB buffer with lower pH can stabilize intact virus particles, but the yield may have a certain impact. ]
Experimental procedure 1. Take 100g of fresh diseased leaves and cut them into 1cm pieces, add 350ml of extraction buffer a, and crush;
2. Filter with heavy gauze;
3. Add TritonX-100 to 3% (more virus particles are obtained, but the yield is low; if carbon tetrachloride is added to 30% TritonX-100 to 2%, a large number of subviral particles can be obtained), 4 Stir at ℃ for 1h;
4. Centrifuge at 5,000 × g for 20 min at 4 ℃;
5. Spread the liquid phase on a 1/3 tube volume of 40% sucrose pad, centrifuge at 40,000 × g for 1.5h, 4 ℃;
6. Suspend the pellet with 2ml buffer;
7. Centrifuge at 3,500 × g for 5min;
8. After diluting the supernatant with buffer, 40,000 × g, 1.5h, 4 ℃, concentrated, and suspended to obtain the crude virus extract. If you want to obtain a relatively pure virus extract, spread it in 20% -50% sucrose On the density gradient, centrifuge at 80,000 × g for 1.5h at 4 ℃;
9. Take 2ml of the centrifuged solution in each tube from top to bottom, and observe under electron microscope. The tube with high virus content is diluted with buffer solution at 40,000 × g, concentrated at 4h for 4h, and the appropriate amount of buffer is suspended.
Precautions Because the virus particles mainly exist in the microtubule bundle, mostly in the phloem, the buffer volume can be reduced to 1/10 and then crushed with a meat grinder.
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