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Merge pull request #2 from stevenweaver/patch-1

Update vRNA extraction.md
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lmoncla committed Apr 9, 2018
2 parents e70bb2c + be2b0e7 commit e6f1569db123476c1a74f019ae51d76aedd758cd
Showing with 2 additions and 2 deletions.
  1. +2 −2 protocols/vRNA extraction.md
@@ -7,7 +7,7 @@ This protocol follows the published protocol for vRNA extraction with the QiAmp
2. Centrifuge at 5,000 x g for 5 minutes at 4°C to pellet host cells. After spin is complete, transfer supernatant to a clean tube and discard tube with pellet.
3. Centrifuge supernatant for 90 minutes at 4°C at maximum speed (14,000 rpm) to concentrate virions in the lower portion of the supernatant.

** About 20 minutes before the spin completes, prepare the buffer AVL with carrier RNA. For each sample you are extracting, you will need 0.56 ml of buffer AVL + 5.6 µl of carrier RNA suspended in buffer AVE. The first time you suspend carrier RNA in buffer AVE, aliquot into tubes and store at -20°C to avoid mµltiple freeze-thaws.
** About 20 minutes before the spin completes, prepare the buffer AVL with carrier RNA. For each sample you are extracting, you will need 0.56 ml of buffer AVL + 5.6 µl of carrier RNA suspended in buffer AVE. The first time you suspend carrier RNA in buffer AVE, aliquot into tubes and store at -20°C to avoid multiple freeze-thaws.

4. Remove ~300 µl of liquid from the top of the supernatant to leave 140-200 µl of total supernatant. Pipet up and down to resuspend viral pellet.

@@ -18,7 +18,7 @@ This protocol follows directly from the manufacturer's recommended protocol.

6. Incubate at room temperature for 10 min. After 10 minutes, lysis is complete and the sample is no longer infectious.

7. Add 560 μl of ethanol (96–100%) to the sample, mix by pµlse-vortexing for 15 seconds, and spin down.
7. Add 560 μl of ethanol (96–100%) to the sample, mix by pulse-vortexing for 15 seconds, and spin down.

8. Transfer 630 μl of the solution from step 5 to the QIAamp Mini column (in a 2 ml collection tube) without wetting the rim. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini column into a clean 2 ml collection tube, and discard the tube containing the filtrate.

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