7. Discard supernatant and add 700 μL of cold 80% ethanol
to wash.
8. Centrifuge for 5 min at 13,000 Â g.
9. Discard supernatant and air-dry the sRNA pellet.
10. Resuspend sRNA pellet in 20 μL of nuclease-free water.
11. Check the quality of the sRNA products using PAGE (see
Note 5). Prepare a 20% polyacrylamide/urea gel by mixing
5 mL of 40% acrylamide solution, 1 mL of 10Â TBE, and
10 mL nuclease-free water. Dissolve 5 g of urea in this mixture
at room temperature and then add 80 μL 10% APS and 10 μL
TEMED immediately before pouring between glass plates set
up on a gel-casting frame, leaving enough room to insert comb
without overflow. Insert comb and allow to polymerize for
about 30 min.
12. When gel is solidified, remove glass plates and gel from the
casting frame, move to gel holder cassette, and place in vertical
electrophoresis chamber. Fill both the inner and outer reservoirs with 0.5Â TBE. Remove comb and rinse wells with 0.5Â
TBE using a micropipette with a fine tip.
13. Take a small aliquot of purified sRNA pellet, heat for 5 min at
95
C, then chill on ice for 5 min, and add appropriate amount
of loading buffer. Load into gel along with 10 bp DNA ladder
and run at 130 V for approximately 2 h.
14. After completion of PAGE, transfer the gel to EtBr staining
solution and incubate on a platform rotator for 5–25 min,
depending on the amount of RNA used. Once staining is
complete, the RNA can be visualized using a UV light source.
15. Use gel to verify the quality of the sRNA preparation and use
Nanodrop spectrophotometry to estimate the concentration.
16. Use immediately or store at À80
C.
3.3 Visualization
of RNA Trafficking into
B. cinerea Mycelium
1. Store B. cinerea B05.0 spores at À80
C, and culture on plates
with solidified malt extract agar (MEA) medium.
2. When petri dish is covered with sporulating fungus, approximately 10 days after plating, pour 5 mL of sterile water onto
the surface and suspend the spores in water using a sterile loop
as aid.
3. Strain the liquid into a 50 mL Falcon tube through a 70 μm
nylon mesh sterile cell strainer.
4. Count spore numbers present in the liquid using a hemocytometer and a benchtop binocular microscope equipped with a
10Â objective.
5. Dilute the liquid with sterile water to 1 Â 10
5 spores/mL.
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Rachael Hamby et al.
to wash.
8. Centrifuge for 5 min at 13,000 Â g.
9. Discard supernatant and air-dry the sRNA pellet.
10. Resuspend sRNA pellet in 20 μL of nuclease-free water.
11. Check the quality of the sRNA products using PAGE (see
Note 5). Prepare a 20% polyacrylamide/urea gel by mixing
5 mL of 40% acrylamide solution, 1 mL of 10Â TBE, and
10 mL nuclease-free water. Dissolve 5 g of urea in this mixture
at room temperature and then add 80 μL 10% APS and 10 μL
TEMED immediately before pouring between glass plates set
up on a gel-casting frame, leaving enough room to insert comb
without overflow. Insert comb and allow to polymerize for
about 30 min.
12. When gel is solidified, remove glass plates and gel from the
casting frame, move to gel holder cassette, and place in vertical
electrophoresis chamber. Fill both the inner and outer reservoirs with 0.5Â TBE. Remove comb and rinse wells with 0.5Â
TBE using a micropipette with a fine tip.
13. Take a small aliquot of purified sRNA pellet, heat for 5 min at
95
C, then chill on ice for 5 min, and add appropriate amount
of loading buffer. Load into gel along with 10 bp DNA ladder
and run at 130 V for approximately 2 h.
14. After completion of PAGE, transfer the gel to EtBr staining
solution and incubate on a platform rotator for 5–25 min,
depending on the amount of RNA used. Once staining is
complete, the RNA can be visualized using a UV light source.
15. Use gel to verify the quality of the sRNA preparation and use
Nanodrop spectrophotometry to estimate the concentration.
16. Use immediately or store at À80
C.
3.3 Visualization
of RNA Trafficking into
B. cinerea Mycelium
1. Store B. cinerea B05.0 spores at À80
C, and culture on plates
with solidified malt extract agar (MEA) medium.
2. When petri dish is covered with sporulating fungus, approximately 10 days after plating, pour 5 mL of sterile water onto
the surface and suspend the spores in water using a sterile loop
as aid.
3. Strain the liquid into a 50 mL Falcon tube through a 70 μm
nylon mesh sterile cell strainer.
4. Count spore numbers present in the liquid using a hemocytometer and a benchtop binocular microscope equipped with a
10Â objective.
5. Dilute the liquid with sterile water to 1 Â 10
5 spores/mL.
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