dsRNA purification (Citrus)
1. Crush 5-7 gr. branch peel in liquid nitrogen.
2. Transfer to a 45ml tube (for Sorval centrifuge) containing:
a. 200 µl β-mercaptoethanol
b. 14 ml STEX2
c. 1.5 ml SDS 10% pH 7.2
d. 18 ml phenol:chloroform 1:1 (phenol is diluted in Tris)
Possibly better to add only half of each component except for STE – add 10 ml.
3. Shake 15 min at room temp.
4. Centrifuge 20 min at room temp., 10,000 RPM.
5. Transfer supernatant to new Sorval tube and add EtOH so it makes up 16.5% of the total volume
(Divide volume of supernatant by 5.24 to calculate volume of EtOH to add).
6. Incubate O/N at 4C, or 1 hr at -20C.
7. Centrifuge 10 min at 7C., 10,000 RPM.
8. Transfer supernatant to 50 ml tube (blue cap) and incubate at room temp. for at least 10 min.
9. In 500 ml Erlenmeyer, mix 2.5 gr CF11 celulose and 60 ml STEX1 solution with 16.5% EtOH, for
each sample.
10. Add a 1 cm radius circle of Whatman filter paper to the bottom of a column and lay the paper
flat. Put the column on a stand over a collection box.
11. With a pipettor, gently add 20 ml of cellulose mixture to each column. Carefully add remaining
mixture to the columns.
12. When all liquid has filtered through, slowly add supernatant from step 8 to column using a
Pasteur pipette. Make sure tip is close to cellulose, but not touching.
13. Wait till all liquid has filtered through. dsRNA should now be bound to cellulose. Wash column
with 30 ml STEX1+16.5% EtOH (15ml+15ml).
14. Elute dsRNA in a Sorval tube with 10ml STEX1 (5ml+5ml). Add carefully.
15. Precipitate dsRNA with 0.1 volumes (1 ml) Sodium acetate 3M pH 5.2 and 2.5 volumes (25ml)
EtOH . Mix gently.
16. Incubate O/N at -20C (or for 1 hr at -80C).
17. Centrifuge 20 min at 7C., 10,000 RPM. Discard supernatant.
18. Wash with 3 ml 75% EtOH
19. Centrifuge as in step 17, discard supernatant.
20. Dry ethanol by inverting tubes on a paper towel and then dry in vacuum for 15 min or until EtOH
has evaporated.
21. Dissolve in 400 µl HPLC water.
22. Vortex well and transfer to 1.5 ml eppendorf tube.
23. Precipitate dsRNA with 0.1 volumes (40 µl) Sodium acetate 3M pH 5.2 and 2.5 volumes (1.2 ml)
EtOH .
24. Incubate O/N at -20C, or 1 hr at -80C. Possible to store at this stage at -20.
25. Centrifuge 20 min at 12,000 RPM at 4C.
26. Dry ethanol as in step 20.
27. Precipitate in 50 µl HPLC water.
28. Nanodrop: Check dsRNA concentration using regular RNA module (factor 40).
29. Store at -20 C.
dsRNA electrophoresis separation
1. Set up electrophoresis gel device. Make sure the frames are water-tight by pouring DW and
making sure the water doesn’t leak out. Then pour the water out and dry the glass surfaces.
2. Prepare 5ml 5% acrylamide gel in 50 ml tube:
0.61 ml acrylamide 40%
0.5 ml 10 X TBE
3.82 ml DDW
65 µl APS 10%
3.4 µl Temed (add last)
3. Mix with tip and use 1 ml pipette to inject liquid between glass frames.
4. Let dry for at least 1 hr, and up to 8 hrs.
5. Pour 1L TBE buffer X1 to cover gel device.
6. Add loading buffer to samples 1:5 - 2 µl loading buffer and 10 µl sample. Load samples slowly
using clean needle. Wash needle 5 times in buffer before and after each load.
7. Run 1 hr at 100V (35 mA).
8. Incubate in 1 mg/mL EtBr for 10 min.
Preparation of solutions:
SDS 10% pH 7.2
100g sodium dodecylsulfate (weigh with dust mask)
900 ml DDW
Stir at 65C in hood
Adjust pH with HCl
Bring to 1L. DO NOT autoclave.
STE (X10)
100 mM Tris HCl pH 6.8
1 M NaCl
10 mM EDTA pH 8
Add DDW to reach desired volume. Autoclave.
STEX1 + 16.5% EtOH
50ml STEX10
82.5ml EtOH
367.5ml DDW
Tris 1M pH 6.8
121.1g Tris base
800 ml DDW
Stir over heat.
Adjust pH with concentrated HCl.
Allow to cool before final pH adjustment.
Adjust to 1 L. Autoclave.
NaCl 5M
292.2g NaCl
800ml DDW
Stir over heat.
Adjust to 1 L. Autoclave.
EDTA 0.5M pH 8
186.1g disodium ethylene diamine tetraacetate . 2H2O
800ml DDW
Stir vigorously on stirrer.
Adjust to pH 8 with NaOH (~20g of pellets)
TBE buffer
1 mM EDTA
90 mM boric acid
90 mM Tris-borat pH 8.3
Loading buffer
0.25% xylene cyanol
0.25% bromo phenol blue
30% glycerol