12. Equipment and supplies for preparing and running a PAGE
gel, including glass plates, casting frame, casting stand and
combs, voltage source, gel holder cassettes, and vertical electrophoresis chamber.
13. N,N,N
0 ,N
0 -tetramethylethylenediamine (TEMED).
14. Urea.
15. 10 bp DNA ladder.
16. 5Â RNA loading buffer.
17. 10% Ammonium persulfate solution (APS).
18. Dry block incubator capable of reaching temperatures of
95
C.
19. EtBr staining solution: 0.5Â TBE + 1 μg/mL ethidium
bromide.
20. Voltage source.
21. UV light source.
22. À20
C Freezer.
23. Benchtop centrifuge capable of speeds up to 13,000 Â g.
2.3 Visualization
of RNA Trafficking into
B. cinerea Mycelium
1. MEA medium: 20 g/L Malt extract, 10 g/L Bacto™ proteose
peptone no. 2, 15 g/L agar.
2. Botrytis cinerea spores stored in À80
C freezer.
3. Nuclease-free water (sterile).
4. Microscope slides (sterile).
5. Petri dish (sterile).
6. 50 mL Falcon tubes (sterile).
7. 70 μm Nylon mesh sterile cell strainer.
8. Benchtop binocular microscope with 10Â objective.
9. Hemocytometer.
10. Micrococcal nuclease.
11. Laser scanning confocal microscope.
12. Sterile hood.
3 Methods
3.1 In Vitro Synthesis
of Fluorescein-Labeled
Double-Stranded
RNAs
1. Prepare DNA template with opposing T7 promoters at the 5
0
ends of each strand by performing PCR on the DNA target of
interest and with primers that each contain the T7 promoter
sequence [5
0 -TAATACGACTCACTATAG-3
0 ] appended to
each 5
0 end. For a 50 μL PCR reaction, add the following
components to a PCR tube on ice (see Note 1) (Step A, Fig. 1):
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Rachael Hamby et al.
gel, including glass plates, casting frame, casting stand and
combs, voltage source, gel holder cassettes, and vertical electrophoresis chamber.
13. N,N,N
0 ,N
0 -tetramethylethylenediamine (TEMED).
14. Urea.
15. 10 bp DNA ladder.
16. 5Â RNA loading buffer.
17. 10% Ammonium persulfate solution (APS).
18. Dry block incubator capable of reaching temperatures of
95
C.
19. EtBr staining solution: 0.5Â TBE + 1 μg/mL ethidium
bromide.
20. Voltage source.
21. UV light source.
22. À20
C Freezer.
23. Benchtop centrifuge capable of speeds up to 13,000 Â g.
2.3 Visualization
of RNA Trafficking into
B. cinerea Mycelium
1. MEA medium: 20 g/L Malt extract, 10 g/L Bacto™ proteose
peptone no. 2, 15 g/L agar.
2. Botrytis cinerea spores stored in À80
C freezer.
3. Nuclease-free water (sterile).
4. Microscope slides (sterile).
5. Petri dish (sterile).
6. 50 mL Falcon tubes (sterile).
7. 70 μm Nylon mesh sterile cell strainer.
8. Benchtop binocular microscope with 10Â objective.
9. Hemocytometer.
10. Micrococcal nuclease.
11. Laser scanning confocal microscope.
12. Sterile hood.
3 Methods
3.1 In Vitro Synthesis
of Fluorescein-Labeled
Double-Stranded
RNAs
1. Prepare DNA template with opposing T7 promoters at the 5
0
ends of each strand by performing PCR on the DNA target of
interest and with primers that each contain the T7 promoter
sequence [5
0 -TAATACGACTCACTATAG-3
0 ] appended to
each 5
0 end. For a 50 μL PCR reaction, add the following
components to a PCR tube on ice (see Note 1) (Step A, Fig. 1):
218
Rachael Hamby et al.