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fixed typos in library prep and RNA extraction protocols

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lmoncla committed Oct 15, 2018
2 parents 9344aab + 1caba1c commit b2f1034b0b1f52896d2e8b42c3fa345fdc468ba1
Showing with 11 additions and 16 deletions.
  1. +9 −14 protocols/Library preparation.md
  2. +2 −2 protocols/vRNA extraction.md
@@ -16,27 +16,22 @@ Library preparation was performed with the Nextera XT DNA Library Prep kit (Illu
6. To each tube, add 7.5 µl of NPM.
7. Being carefµl not to touch the caps, add 2.5 µl of the i5 indices to each tube.
7. Being careful not to touch the caps, add 2.5 µl of the i5 indices to each tube.
8. Add 2.5 µl of the i7 indices to each tube. Pipette up and down 5 times to mix.
9. Place tubes in a thermocycler and run the following limited-cycle PCR program.
Ensure that the thermocycler lid is heated during the incubation. (total volume = 25 µl)
72°C for 3 minutes
95°C for 30 seconds
12 cycles of:
95°C for 10 seconds
55°C for 30 seconds
72°C for 30 seconds
72°C for 5 minutes
Hold at 10°C
1. 72°C for 3 minutes
2. 95°C for 30 seconds
3. 12 cycles of:
1. 95°C for 10 seconds
2. 55°C for 30 seconds
3. 72°C for 30 seconds
4. 72°C for 5 minutes
5. Hold at 10°C
* After the PCR step is complete, reaction products can be stored at 4°C for up to 2 days.
@@ -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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