RNA extraction (bioreba)
Prior to start:
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Add ethanol to newly opened W1/W2.
Prepare full ice bucket
1. Prepare 1.5ml tubes with 200 µl binding buffer (VB).
Virus with polyA (GVA, leafroll virus)  use VB without polyA
Virus without polyA (CTV)  use VB including polyA.
2. Prepare empty 2ml tubes.
3. Prepare empty bioreba bags
4. Add 5ml buffer to each bag.
Grapevine (‫ )גפן‬ orange buffer diluted 1:5 with cold DW (10 ml buffer, 40 ml DW)
Citrus  green buffer diluted 1:10 with cold DW (5 ml buffer, 45 ml DW)
5. Add a pinch (‫ )קומץ‬of sodium bisulfide to the bag.
6. Add 1g plant tissue to front of bag, and incubate on ice.
7. Crush leaves in bag.
8. Using a Pasteur pipet, remove liquid from back of bag to 2ml tubes. Incubate tubes on ice.
9. Centrifuge 1-3 min at 13,000 RPM.
10. Transfer 400 µl supernatant to tubes with VB.
11. Vortex 5 sec.
12. Incubate 10 min room temp.
13. Add 100 µl isopropanol and invert tubes (no need to centrifuge). Change gloves.
*At this point it is possible to keep tubes at -20C for several hours.
14. Transfer liquid onto binding column.
15. Centrifuge 1 min 8000 RPM. Make sure liquid completely passes through.
16. Discard liquid and return column to new 2 ml collection tube.
17. Incubate elution buffer (EB) at 60C.
18. Add 500 µl W1 to the column.
19. Centrifuge 1 min 8000 RPM. Discard liquid.
20. Add 500 µl W2 to the column and centrifuge 1 min 8000 RPM. Discard liquid.
21. Centrifuge 1 min at 13,000 RPM to remove W2 completely.
22. Transfer column to final 1.5ml tube. Add 30-50 µl heated EB to tube center.
23. Incubate 5 min at room temp. Note: tubes do not close.
24. Centrifuge 1 min 8000 RPM.
Note: it is possible to carry out steps 22-24 twice. Each time add only 15-25 µl EB. This may help
elution.
25. Discard column not tube!