Isolation and Visualization of Rotavirus Genome via Gel Electrophoresis

Take a debris-free cell lysate containing rotavirus.
Add a denaturing salt solution and vortex. The salt denatures host proteins, inactivates RNases, and destabilizes the viral capsid.
Add chloroform and vortex to rupture the viral capsid, releasing viral RNA segments.
Centrifuge the suspension. The RNA collects in the aqueous phase, while denatured capsid proteins accumulate at the interface.
Transfer the aqueous phase and add isopropyl alcohol, which lowers nucleic acid solubility and precipitates RNA.
Centrifuge to separate RNA. Discard the supernatant, and add ethanol.
Centrifuge to pellet RNA and discard the supernatant.
Air-dry to remove traces of ethanol, then dissolve RNA in nuclease-free water to preserve RNA integrity.
Mix RNA with loading buffer and load it onto a polyacrylamide gel.
Run electrophoresis for size-based separation of viral RNA segments.
Stain the RNA with an RNA-intercalating dye.
Under ultraviolet light, eleven distinct bands confirm recovery of the rotavirus genome.
Add 600 microliters of clarified infected cell lysates and 400 microliters of guanidinium thiocyanate into 1.5 milliliter microcentrifuge tubes. Vortex them for 30 seconds and incubate them at room temperature for 5 minutes.
Then add 200 microliters of chloroform. Vortex the solution for 30 seconds and incubate it for 3 minutes. After a five-minute microcentrifugation at 13,000 times g and 4 degrees Celsius, transfer 550 microliters of the upper aqueous phase into a fresh tube.
Add two volumes of cold isopropyl alcohol and invert the tube four to six times. Incubate the sample at room temperature for 10 minutes. Then centrifuge it for 10 minutes at 13,000 times g and 4 degrees Celsius.
Discard the supernatant, leaving the RNA pellet. Wash the RNA by adding 1 milliliter of 75% ethanol to the tube, inverting it once, and centrifuging it for 5 minutes at 7,500 times g and 4 degrees Celsius. After carefully removing the ethanol, allow RNA to air dry for 5 to 10 minutes.
Then dissolve the pellet in 15 microliters of nuclease free water. Add 2 microliters of 6X DNA loading buffer to 10 microliters of the dissolved RNA sample. Load it onto a pre-cast 10% polyacrylamide mini gel.
Resolve the RNAs by electrophoresis in Tris-glycine running buffer for 2 hours under a constant 16 milliampere current. Once the gel run is complete, soak the gel for 5 to 10 minutes in water containing 1 microgram per milliliter ethidium bromide and detect the rotavirus genome segments with a UV transilluminator.