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Vacuum Protocol: Purification of Viral Nucleic acid from Plasma or Serum

互联网2013-11-13

925

实验试剂

Regents supplied by user

1. Ethanol -approximately 0.3 ml per sample.

实验设备

Equipments supplied by user

1. Tabletop microcentrifuge and sterile 1.5 ml tubes

2. Vacuum Manifold

3. Water bath - set to 37°C

实验步骤

1. Transfer 25 μl OB Protease into a sterile microcentrifuge tube.

2. Add 200 μl Plasmid or Serum into the tube and bring the volume up to 225 ul with DEPC treated water.

3. Add 225 ul Buffer TNA and 5.6 μl Carrier RNA. Mix thoroughly by vortexing.

4. Incubate sample at 45°C for 10 min. Briefly vortex the tube once during incubation.

5. Add 280 ul of isopropanol and mix thoroughly by vortexing. Incubate at room temperature for 5 minutes.

6. Insert the HiBind® MicroElute Viral Column into the vacuum manifold. Carefully apply the lysate to HiBind® MicroElute Viral column. Turn on the vacuum source to draw all liquid through the column. Turn off the vacuum.

Note: If the lysate has difficulty to pass through the column at this stage. Place the column into a collection tube (supplied). Close the lid and centrifuge at 8,000 x g for 5 minutes or until all liquid pass through the column. Place the column into another collection tube (supplied) and continue step 7 of the spin protocol.

7. Pipet 500 ul of Buffer VHB into the column. Turn on the vacuum source to draw all liquid through the column. Turn off the vacuum.

8. Wash the column by pipetting 500 ul of RWB Wash Buffer diluted with ethanol into the column. Turn on the vacuum source to draw all liquid through the column. Turn off the vacuum.

9. Close the lid of HiBind® MicroElute Viral column, remove it from the vacuum manifold. Insert the column into a collection tube (supplied) and centrifuge at 15,000 x g for 2 minute to completely dry the column.

10. Place the column into a sterile 1.5 ml microfuge tube and add 15-50 ul of DEPC-Treated water. Allow tubes to sit for 5 min at room temperature.

11. To elute nucleic acid from the column, centrifuge at 8,000 x g for 1 min. Retain flow-through containing the nucleic acid. Place column into a second 1.5 ml tube.

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