Protocol for Manual Isolation of Viral DNA/RNA in Microfuge Tubes (1.5 or 2.0 ml)

Important Notes Before You Begin

• Review Reagent Preparation section.
• Store Proteinase K at –20°C upon receipt.
• Prepare Monarch Carrier RNA based on kit size used: Add 125 µl (NEB #T4010S) or 750 µl (NEB #T4010L/X) nuclease-free water, invert or pipette to mix, and transfer to an RNase-free microfuge tube. Keep on ice. Prepare single-use aliquots and store at –20°C. Avoid multiple freeze-thaw cycles.
• Prepare 80% ethanol: 80% ethanol should be prepared fresh using 100% absolute ethanol (user supplied) and nuclease-free water (user supplied). Prepare 1 ml of 80% ethanol per reaction and add overage.
• Perform all steps at room temperature unless directed otherwise.

Starting Material Notes

This protocol has been optimized for use with 200 μl saliva or a respiratory swab sample collected in viral transport media (VTM). For samples < 200 μl, the sample volume should be adjusted to 200 μl with VTM or PBS before processing.

Part I. Buffer Preparation

1. Prepare fresh Viral DNA/RNA Wash Buffer in a user-supplied tube or bottle (free of nucleases) according to the table. Add components in order, as listed. Prepare up to 15% excess to ensure a sufficient volume is available for each reaction.
2. Prepare Lysis Buffer Bead Mix immediately before use, according to the table.
   1. Vortex magnetic beads to form a homogeneous solution before use.
   2. Add components in order, as listed.
   3. For a master mix, prepare up to 15% excess to ensure a sufficient volume of buffer/bead mix is available for each reaction.
   4. Store Lysis Buffer Bead Mix at room temperature. Periodically invert or vortex to keep beads in suspension.

Part II. Sample Lysis

1. Add 5 μl Proteinase K to each 1.5 ml microfuge tube.
2. Add 200 μl sample (e.g., saliva or nasal swab in VTM) to each tube, and pipette thoroughly to mix.
3. Incubate tubes at room temperature for 15 minutes.
4. Gently vortex Lysis Buffer Bead Mix before adding 421 μl to each sample tube. Pipette gently but thoroughly to mix.

Part III. Viral Nucleic Acid Purification (Bind, Wash, Elute)

Bind nucleic acid to beads

1. Mix samples in a thermal mixer for 5 minutes at 2000 rpm.
2. Spin tubes briefly in a benchtop mini centrifuge to collect liquid at the bottom of the tube.
3. Place tubes on magnet for 3 minutes.
4. With tubes on the magnet, carefully remove and discard the supernatant.

Wash beads

5. Remove tubes from the magnet and add 500 µl Viral DNA/RNA Wash Buffer.
6. Mix samples in a thermal mixer for 1 minute at 2000 rpm (or vortex mix, 5 seconds).
7. Spin tubes briefly in a benchtop mini centrifuge.
8. Place tubes on the magnet for 3 minutes.
9. With tubes on the magnet, carefully remove and discard the supernatant.
10. Repeat wash steps 5–9, washing beads with 500 µl 80% ethanol.
11. Repeat wash steps 5–9 for a second wash with 500 µl 80% ethanol.
12. Spin tubes briefly in a benchtop mini centrifuge.
13. Place tubes on the magnet for 3 minutes.
14. With tubes on the magnet, carefully remove and discard any residual supernatant.

Dry beads

15. Air-dry beads for 5 minutes. If visible liquid droplets remain in the tube or on the bead pellet, extend the drying time up to 10 minutes. (During this bead drying step, set the thermal mixer to 65°C).

Elute nucleic acid

16. Remove tubes from the magnet and add 33–100 μl nuclease-free water.
17. Mix samples in a thermal mixer at 65°C for 5 minutes at 2000 rpm.
18. Place tubes on the magnet for 5 minutes.
19. Transfer the supernatant (i.e., eluted nucleic acid) to a nuclease-free microfuge tube.
20. Place eluate on ice for immediate use or at –80°C for long-term storage.