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Total RNA Isolation: 100 l Viral Sample From Cultured-Cell Supernatant Containing Virus


AquaPure RNA Isolation Kit
Catalog #732-6370
Yield: Dependent on sample and RNA expression levels


Method
Sample Collection and Handling
Samples may be fresh or frozen. Collect fresh virus and isolate RNA as quickly as possible; store at 18C for not more than 24 hr. For long-term storage, store at -70 to -80C.

Cell Lysis
1. Add 500 l cell lysis solution to a 1.5 ml microfuge tube that contains 100 l virus from cultured-cell supernatant.

2. Cap the tube and vortex for 5 sec to mix thoroughly.

3. Heat sample to 65C for 5 min to complete lysis. Cool sample to room temperature. (This step is optional when testing for certain viruses such as hepatitis C.)

Protein-DNA Precipitation
1. Add 200 l protein-DNA precipitation solution to the viral cell lysate.

2. Invert capped tube gently 10 times and place tube into an ice bath for 5 min.

3. Centrifuge at 13,00016,000 x g for 3 min. The precipitated proteins and DNA will form a tight pellet.

RNA Precipitation
1. Leaving behind the precipitated protein-DNA pellet, pour the supernatant containing the RNA into a clean 1.5 ml microfuge tube containing 600 l 100% isopropanol (2-propanol). If RNA yield is expected to be low (<1 mg), add glycogen as a carrier to the isopropanol. We recommend adding 1 l of glycogen solution (20 mg/ml) per 600 l isopropanol.

2. Cap the tube and mix the sample by inverting gently 50 times.

3. Centrifuge at 13,00016,000 x g for 3 min; depending on yield, the RNA may or may not be visible as a small, translucent pellet.

4. Pour off supernatant and drai n tube on clean absorbent paper. Add 600 l 70% ethanol and invert the capped tube several times to wash the RNA pellet.

5. Centrifuge at 13,00016,000 x g for 1 min. Carefully pour off the ethanol.

6. Invert and drain the tube on clean absorbent paper and allow to air-dry 1015 min.

RNA Hydration
1. Add 20 l RNA hydration solution (20 l will give a concentration of 50 g/ml if the total yield is 1 g RNA).

2. Allow RNA to rehydrate on ice for at least 30 min. Store purified RNA sample at 70 to 80C until use.

3. Before use, vortex sample vigorously for 5 sec and pulsespin. Pipet sample up and down several times to ensure adequate mixing.



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