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Sample Buffer G-AV Buffer G-BV Buffer G-CV	110 330 198 220	120 360 216 240	130 390 234 260	140 420 252 280	150 450 270 300	160 480 290 320

1. Collect 160 l of sample and apply in 1.5-ml Microfuge Tube.

2. Add 0.48 ml of Buffer G-AV pre-chilled at 4?, vortex to mix well, chill on ice for 5 min.

3. Add 0.29 ml of Buffer G-BV, mix well.

4. Transfer the mixture in Filter (placed in 2-ml Microfuge Tube). Centrifuge at 12000×g for 30 s.

5. Place the Filter in a new 2-ml Microfuge Tube. Discard the upper-phase, and transfer the lower-phase to the Filter. Centrifuge at 12000×g for 30 s.

* During transferring the lower-phase, complete removal of the upper-phase is not necessary since occasionally aspirated upper-phase will quickly shift onto the top of liquid in the Tip and is easy to be discarded.

6. Discard the Filter, add 0.32 ml of Buffer G-CV in the filtrate and mix well.

7. Place Nucleic acid-prep Tube in a new 2-ml Microfuge Tube. Transfer the mixture in step 6 to Nucleic acid-prep Tube. Centrifuge at 1500×g for 1 min.

* If there is solution remaining in the tube, increase the centrifugation speed and spin once again.

8. Discard the flow-through in the 2-ml microfuge tube. Place the Nucleic acid-prep Tube back in the 2-ml Microfuge Tube. Add 0.5 ml of Buffer W1 to the Nucleic acid-prep Tube and centrifuge at 1500×g for 1 min.

* If there is solution remaining in the tube, increase the centrifugation speed and spin once again.

9. Discard the flow-through and place Nucleic acid-prep Tube back in the 2-ml Microfuge Tube. Add 0.7 ml of Buffer W2, and centrifuge at 1500×g for 1 min. Discard the flow-though and wash once again with 0.7 ml of Buffer W2.

10. Transfer the Nucleic acid-prep Tube in a 1.5-ml Microfuge Tube and centrifuge at 12000×g for 1 min to remove residual Buffer W2.

11. Transfer the Nucleic acid-prep Tube in a new 1.5-ml Microfuge Tube. To elute DNA, add 30 l of water or the Eluent to the center of the silica membrane, Centrifuge at 12000×g for 30s.

* The purified viral RNA should be used for RT-PCR analysis immediately, or store at -20°C

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