15. Mix reaction by flicking the tube or pipetting the mixture up
and down, and then briefly spin to collect the mixture at the
bottom of the tube.
16. Incubate at 37
C for 2 h.
17. Stop the reaction by adding 2 μL of 0.2 M EDTA, pH 8.0.
18. To purify the dsRNA, add 2.5 volumes of 100% ethanol and
0.1 volumes of 3 M sodium acetate (pH 5.5) and precipitate at
À20
C overnight.
19. Centrifuge at 4
C for 15 min at 13,000 Â g.
20. Discard supernatant and add 700 μL of cold 75% ethanol
to wash.
21. Centrifuge for 5 min at 13,000 Â g.
22. Discard supernatant and resuspend dsRNA pellet in 30 μL of
nuclease-free water.
23. Run a small aliquot on an agarose gel, as described above, to
check the quality of the product and use Nanodrop spectrophotometry to estimate the concentration.
24. Use immediately or store at À80
C.
3.2 Preparation
of Fluorescent sRNAs
1. Use the fluorescent dsRNA synthesized in step 1 and combine
the following in a microcentrifuge tube on ice to produce a
100 μL reaction (Step C, Fig. 1):
Reagent
Volume (μL)
10 μg dsRNA
x
10Â ShortCut
® Reaction Buffer
10
ShortCut
® RNase III
10
200 mM MnCl 2
10
Nuclease-free water to a final volume of 100 μL
x
2. Gently mix the volume with a pipette and incubate at 37
C for
20 min.
3. To stop the reaction, add 10 μL of 250 mM EDTA (see
Note 3).
4. To precipitate the digestion product, transfer it to a 1.5 mL
tube and add 2 μL RNase-free glycogen, 3 volumes of cold
100% ethanol, and 0.1 volumes of 3 M sodium acetate
(pH 5.5) (see Note 4).
5. Precipitate at À20
C overnight.
6. Centrifuge at 4
C for 15 min at 13,000 Â g.
Visualizing Fungal RNA Uptake
221