TITLE: NEB #T4010 Monarch Mag Viral DNA/ RNA Extraction Kit Protocol for KingFisher Flex Automated Isolation of Viral DNA/RNA from Wastewater Samples Following Ceres Nanotrap Enrichment
AUTHORS: Juliet Bonnevie

[DESCRIPTION]
The Monarch Mag Viral DNA/RNA Extraction Kit provides a rapid and reliable magnetic bead-based process for extracting viral nucleic acids from saliva and respiratory swab samples. The kit combines the efficiency of silica-based nucleic acid purification with the ease of use of magnetic beads. Manual and automated workflows allow samples to be processed in microfuge tubes or 96-well plates. Kit sizes align to 96-well formats (100 preps, 600 preps, and 1800 preps), and the protocol is compatible with high throughput automation on a variety of platforms, including the KingFisher Flex magnetic particle processor and Agilent Bravo and MGISP liquid handler platforms.

[BEFORE_START]
Important Notes Before You Begin
Review Reagent Preparation section in the manual on NEB.com.
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 1 ml of 80% ethanol per reaction and add overage.
Perform all steps at room temperature unless directed otherwise.

Required plastics
KingFisher 96-deep well plates, v-bottom, (2.0 ml), Catalog # 95040450
KingFisher 96 microplate (200 μl), Catalog # 97002540
KingFisher 96 deep-well tip comb and plate, Catalog # 97002820

Starting Material Notes
This protocol has been optimized for use with wastewater samples that have been pre-processed with Ceres Nanotrap Particles.

[STEPS]
SECTION: Part I. Prepare the KingFisher Flex Instrument
1. Ensure the instrument is equipped with the KingFisher Flex 96 Deep Well head and the KingFisher Flex 96 heating block.
IMPORTANT: The heat block must be compatible with the KingFisher 96 microplate (200 μl).
SECTION: Part I. Prepare the KingFisher Flex Instrument
2. Ensure the MagMAX Pathogen RNA/DNA (High Volume) program is loaded onto the instrument’s connected computer and that the program has been modified to perform three 500 μl wash steps, a 2-minute bead drying step, and a 33–100 μl elution.
SECTION: Part I. Prepare the KingFisher Flex Instrument
3. Enter sample, wash, and elution volumes into the program.
SECTION: Part I. Prepare the KingFisher Flex Instrument
4. Select plate sizes for the run: KingFisher 96-deep well plates (2.0 ml) for sample and wash plates; KingFisher 96 microplate (200 μl) for elution.
SECTION: Part II. Buffer Preparation
5. 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.
SECTION: Part II. Buffer Preparation
6. Prepare Lysis Buffer Bead Mix immediately before use, according to the table.
a. Vortex magnetic beads to form a homogeneous solution before use.
b. Add components in order, as listed.
c. For a master mix, prepare up to 15% excess to ensure a sufficient volume of buffer/bead mix is available for each reaction.
d. Store Lysis Buffer Bead Mix at room temperature. Periodically invert or vortex to keep beads in suspension.

Viral DNA/RNA Wash Buffer:
 

Lysis Buffer Bead Mix
 

SECTION: Part III. Prepare Wash and Elution Plates
7. Aliquot 500 μl Viral DNA/RNA Wash buffer to wells in a 96-well deep well plate.
	
SECTION: Part III. Prepare Wash and Elution Plates
8. Aliquot 500 μl 80% ethanol to wells in each of two 96-well deep well plates.
SECTION: Part III. Prepare Wash and Elution Plates
9. Aliquot 33–100 μl nuclease-free Water to wells in a 96-well microplate.
SECTION: Part III. Prepare Wash and Elution Plates
10. Seal plates with an adhesive film until ready to use.
 

SECTION: Part IV. Elute target microbes from the Nanotrap pellet
11. These steps will begin at the end of enrichment, specifically where Ceres protocol adds a Lysis Buffer. For example, for Protocol APP-042, you would follow the Nanotrap protocol till Step 11 and perform the steps below instead of Step 12.

Add 100 μl Monarch StabiLyse DNA/RNA Buffer to the Nanotrap Particle pellet, pipette to resuspend the pellet.
SECTION: Part IV. Elute target microbes from the Nanotrap pellet
12. Incubate for 10 mins at room temperature.
SECTION: Part IV. Elute target microbes from the Nanotrap pellet
13. Use a magnetic rack that is compatible with sample tubes to separate Nanotrap Particles from the sample.
SECTION: Part IV. Elute target microbes from the Nanotrap pellet
14. Transfer the supernatant to a deepwell plate compatible with processing on KingFisher.
SECTION: Part IV. Elute target microbes from the Nanotrap pellet
15. Sample Lysis (Sample Plate)

Add 100 μl nuclease-free water to the Nanotrap enriched sample from Step 14 above.
SECTION: Part IV. Elute target microbes from the Nanotrap pellet
16. 111 Add 5 ul Proteinase K.

 

SECTION: Part IV. Elute target microbes from the Nanotrap pellet
17. Pipette mix or use a thermomixer for 30 seconds after sealing the plate.
SECTION: Part IV. Elute target microbes from the Nanotrap pellet
18. Incubate for 15 min at room temperature.
SECTION: Part IV. Elute target microbes from the Nanotrap pellet
19. Gently vortex the prepared Lysis Buffer Bead Mix and add 421 μl to each sample well.
SECTION: Part IV. Elute target microbes from the Nanotrap pellet
20. Seal plate with adhesive film until ready to load onto the KingFisher Flex instrument.
SECTION: Part V. Viral Nucleic Acid Purification (Bind, Wash, Elute)
21. Carefully remove adhesive film from sample, wash, and elution plates.
SECTION: Part V. Viral Nucleic Acid Purification (Bind, Wash, Elute)
22. Load sample, wash, elution plates, tip comb and plate, into the appropriate positions on the KingFisher Flex worktable.
SECTION: Part V. Viral Nucleic Acid Purification (Bind, Wash, Elute)
23. Run the modified MagMAX program.
SECTION: Part V. Viral Nucleic Acid Purification (Bind, Wash, Elute)
24. Upon completion of the run, seal the elution plate with adhesive film and place on ice for immediate use or freeze for storage.