5. Thermal block for 1.5 mL tubes.
6. Hybridization tube.
7. Hybridization oven.
8. Hybridization buffer (e.g., PerfectHyb™ Plus, SigmaAldrich).
9. DNA oligonucleotide complementary to viral sequence of
interest, with a calculated melting temperature (in water) of
around 55–60
C. Generally, this oligonucleotide should be
20–25 nucleotides long. Crucially, this oligonucleotide should
give no signal when used as probe in Northern blot on virusfree samples.
10. Polynucleotide kinase (PNK) labeling kit.
11.
32
P-γATP, along with all materials, protective gear, and authorizations required to handle radioactive isotopes and to dispose
of radioactive waste.
12. Filter tips.
13. Paper towel cut into small squares of 5 Â 5 cm and folded
twice.
14. 20% SDS solution.
15. Wash buffer: 2% SDS, 2Â SSC.
16. Tweezers.
17. Transparent plastic envelope.
18. Radioactivity-detection equipment of choice: Cassette with
radiation-reflecting screens and autoradiographic film, or
phosphor-imager screen and detector.
2.5 Western Blot
1. 4Â Protein-loading buffer: 250 mM Tris–HCl pH 6.8, 40%
glycerol, 8% SDS, 20% (v/v) 2-ß-mercaptoethanol.
2. Protein resuspension buffer: 62.3 mM Tris–HCl pH 8, 10%
glycerol, 3% SDS.
3. Heating block for 1.5 mL tubes.
4. Tabletop centrifuge capable of providing 16,000 Â g at 4
C.
5. Acetone (ice cold).
6. 80% Acetone (ice cold).
7. SDS-PAGE gel-casting system.
8. Glass plates for SDS-PAGE gels.
9. Well combs for SDS-PAGE gels.
10. Acrylamide/bis-acrylamide 37.5/1.
11. Resolving buffer: 1.1 M Tris–HCl, 0.4% SDS, pH 8.8.
12. Stacking buffer: 0.6 M Tris–HCl, 0.4% SDS, pH 6.8.
13. 10% Ammonium persulfate (APS).
Pull-Down of Viral Double-Stranded RNA
311
6. Hybridization tube.
7. Hybridization oven.
8. Hybridization buffer (e.g., PerfectHyb™ Plus, SigmaAldrich).
9. DNA oligonucleotide complementary to viral sequence of
interest, with a calculated melting temperature (in water) of
around 55–60
C. Generally, this oligonucleotide should be
20–25 nucleotides long. Crucially, this oligonucleotide should
give no signal when used as probe in Northern blot on virusfree samples.
10. Polynucleotide kinase (PNK) labeling kit.
11.
32
P-γATP, along with all materials, protective gear, and authorizations required to handle radioactive isotopes and to dispose
of radioactive waste.
12. Filter tips.
13. Paper towel cut into small squares of 5 Â 5 cm and folded
twice.
14. 20% SDS solution.
15. Wash buffer: 2% SDS, 2Â SSC.
16. Tweezers.
17. Transparent plastic envelope.
18. Radioactivity-detection equipment of choice: Cassette with
radiation-reflecting screens and autoradiographic film, or
phosphor-imager screen and detector.
2.5 Western Blot
1. 4Â Protein-loading buffer: 250 mM Tris–HCl pH 6.8, 40%
glycerol, 8% SDS, 20% (v/v) 2-ß-mercaptoethanol.
2. Protein resuspension buffer: 62.3 mM Tris–HCl pH 8, 10%
glycerol, 3% SDS.
3. Heating block for 1.5 mL tubes.
4. Tabletop centrifuge capable of providing 16,000 Â g at 4
C.
5. Acetone (ice cold).
6. 80% Acetone (ice cold).
7. SDS-PAGE gel-casting system.
8. Glass plates for SDS-PAGE gels.
9. Well combs for SDS-PAGE gels.
10. Acrylamide/bis-acrylamide 37.5/1.
11. Resolving buffer: 1.1 M Tris–HCl, 0.4% SDS, pH 8.8.
12. Stacking buffer: 0.6 M Tris–HCl, 0.4% SDS, pH 6.8.
13. 10% Ammonium persulfate (APS).
Pull-Down of Viral Double-Stranded RNA
311