3.2.1. Polyacrylamide Gel Electrophoresis
1. Vertical gel electrophoresis apparatus (Life Technologies, Paisley, UK).
2. Initially wash the glass plates in 1% (w/v) SDS, rinse in deionized water, and dry the plates completely.
3. Prepare 70 mL of a 10% (w/v) polyacrylamide gel by initially preparing the resolving gel (see Note 5).
4. Add (in order) 23.28 mL distilled water, 13.2 mL 38% (w/v) acrylamide mix, 12.5 mL 1.5 MTris-HCl, pH 8.8, 1.5 mL 10% (w/v) SDS, 0.5 mL 10% (w/v) ammonium persulfate, and 0.02 mL TEMED.
5. Pour this mixture immediately into the gel cast leaving sufficient space for the stacking gel (approx 2 cm).
6. Overlay the acrylamide solution with isobutanol (see Note 6) and allow sufficient time for polymerization to occur (approx 30 min).
7. Pour off the overlay and wash the gel surface several times with deionized water.
8. Prepare 20 mL of the stacking gel (see Note 5) by adding (in order) 11.78 mL distilled water, 5.3 mL 38% (w/v) acrylamide mix, 2.5 mL 1 M Tris-HCl, pH 6.8, 0.2 mL 10% (w/v) SDS, 0.2 mL 10% (w/v) ammonium persulfate, and 0.02 mL TEMED. Mix the components and pour directly onto the resolving gel.
9. Insert a Teflon comb into the stacking gel and continue polymerization for a further 30 min at room temperature.
10. Remove the comb and wash the preformed wells with deionized water to remove any unpolymerized acrylamide.
11. Place the gel into the electrophoresis apparatus and add electrophoresis buffer to the top and bottom buffer reservoirs.
12. Prepare samples for electrophoresis by mixing 20 ^L of sample RNA and 20 ^l of gel loading dye (see Note 7).
13. Load all of this mixture directly onto the gel and perform electrophoresis for 16 h at 25 mA using 1X Tris-glycine, pH 8.3 buffer.
3.2.2. Staining and Detection of dsRNA Segments in Polyacrylamide Gels
1. Following electrophoresis, fix the gel for 30 min in a 10% (v/v) ethanol-acetic acid solution.
2. Silver stain with 0.011 M silver nitrate for 2 h then briefly (1 min) rinse in deionized water.
3. Transfer the gel to the developing solution (buffer 2) for 20 min, or until the RNA segments are clearly visible (not longer than 45 min).
4. Increase the intensity/visibility of the segments by immersing the gel in a solution of freshly prepared 0.07 M sodium carbonate for 10 min.
5. Visualize the electrophoretic patterns (electropherotypes) over a white light box.
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