Protocol Guidance for Automated Isolation of Viral DNA/RNA in Deep Well Plates

Refer to the product webpage for the most up-to-date information on additional guidance and protocols for other automation/liquid handling instruments.

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% ethanol (user-supplied) and nuclease-free water (user supplied). Prepare 320 μl 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. Instrument Preparation

1. Ensure the automation instrument is equipped with the appropriate hardware (e.g., magnet, shaker, heat block).
2. Load an appropriate script onto the instrument that aligns with sample, wash, elution volumes, and sample processing steps
   (e.g., sample/bead mixing, bead collection, supernatant removal, wash steps, bead drying, and heated elution).
3. Ensure instrument-compatible plastics are used and mixing speeds are compatible with liquid volumes.

Part II. 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.
3. Vortex magnetic beads to form a homogeneous solution before use.
4. Add components in order, as listed.
5. For a master mix, prepare up to 15% excess to ensure a sufficient volume of buffer/bead mix is available for each reaction.
6. Store Lysis Buffer Bead mix at room temperature. Periodically invert or vortex to keep beads in suspension.

Part III. Sample Lysis

1. Add 5 μl Proteinase K to plate wells (1.0 ml, 96-well deep well plate).
2. Add 200 μl sample (e.g., saliva or nasal swab in VTM), and pipette thoroughly to mix.
3. Seal the plate with an adhesive film and incubate at room temperature for 15 minutes.
4. Carefully remove the film.
5. Gently vortex Lysis Buffer Bead Mix and add 421 μl to each well. Pipette gently but thoroughly to mix.

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

Bind nucleic acid to beads

1. Mix sample plate at ~1000 rpm for 5 minutes.
2. Collect beads on magnet for 3 minutes. Remove supernatant.

Wash beads

3. Add 160 μl Viral DNA/RNA Wash Buffer to beads.
4. Mix at ~700 rpm for 2 minutes.
5. Collect beads on magnet for 3 minutes.
6. Remove supernatant.
7. Repeat wash steps 3–6 with 160 μl 80% ethanol.
8. Repeat wash steps 3–6 for a second wash with 160 μl 80% ethanol.

Dry beads:

9. Dry beads for 30 seconds – 1 minute.

Elute nucleic acid:

10. Add 33–100 μl nuclease-free water to beads.
11. Incubate plate at 65°C with mixing for 5 minutes.
12. Collect beads on magnet for 6 minutes.
13. Transfer eluate to 96-well plate.
14. Place eluted nucleic acid on ice for immediate use or at –80°C for storage.