W-2 WATER PROCESSING
REDI-NET Consortium
Disclaimer
This work is supported by the US Army Medical Research and Development Command under Contract No.W81XWH-21-C-0001, W81XWH-22-C-0093 and HT9425-23-C-0059. The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army or Navy position, policy or decision unless so designated by other documentation.
Abstract
This protocol details about water processing.
Before start
BEFORE START
- Make sure the feeding tube and PTFE sinker for SolVac Filter are properly clean by using 70% ethanol and allowed to air dry.
- For the samples stored at
4°C, pour three250mLsamples from the same sampling location into a sterile 1190 mL sample bag. - If using a frozen water sample, fully thaw them at room temperature. For the frozen sample in a plastic sample bag, wipe the bag surface with 70% ethanol to remove dusts and sanitize the surface. Source three bags of
250mLwater samples from the same sampling location in a new 1190 ml sterile sample bag, then put the bagged samples in a suitable-sized container for defrosting. After samples are fully defrosted, pour the water samples into the 1190 ml outer bag and discard the original 250 ml sample bags. Hold the whole bag in a 1 L beaker. - Prepare 40% PEG-8000 solution for microbe aggregation. Check Appendix 3 for the recipe.
- Pre-cool the Bullet Blender by adding dry ice into the cooling compartment and running the cooling program.
- Clean the work surfaces with RNaseZap, then wipe the surfaces with 70% molecular biology grade ethanol to remove additional contaminants.
- Transfer 0.1 mm zirconium oxide beads (2 spoons, Appendix 4 ) to Clear RINO brand 1.5 ml screw-cap microcentrifuge tubes.
- For the first time use of IndiMag pathogen kit, add 100% ethanol to Buffer AW1 and AW2, and add 100% Isopropanol to ACB as indicated on the bottles.
- Buffer ATL may form precipitates upon storage. If necessary, warm to
56°Cuntil the precipitates have fully dissolved. Prepare buffer ATL-DX: add100µLReagent DX to15mLBuffer ATL. If smaller amounts are needed, transfer1.5mLof Buffer ATL into a sterile 2 ml vial and add10µLReagent DX. Mix well, after the addition of Reagent DX. After preparation, the mixture is stable for 6 months atRoom temperature(15-25°C). - MagAttract Suspension G from the IndiMag pathogen kit needs to be vortexed thoroughly for
0h 3m 0s(before first use) or 1 minute (before subsequent uses) to ensure that the magnetic silica particles are fully resuspended. - Binding beads need to be vortexed thoroughly before each use.
- Prepare a few 15 ml or 50 ml conical centrifuge tubes with nuclease-free water for preparing TNA elution in KingFisher Flex or KingFisher Duo Prime to avoid cross-contamination.
Steps
1. VACUUM PUMP SET UP
Wipe the surfaces with 70% ethanol to remove contaminants.
Use tubing to connect a 3 liter Medi-Vac Canister with vacuum pump through the vacuum outlet on the lid.
Connect tubing with the 3 liter Medi-Vac Canister through the inlet on the lid. Close unused inlets. Turn on the pump to test the vacuum suction by feeling the airflow.
2. WATER SAMPLE PRE-FILTRATION
Wipe the housing base with 70% ethanol and let ethanol air dry (Vacuum pump can be turned on to help drying).
Clip a thumb clamp on the feeding tubing.
Connect upper housing, feeding tubing, and PTFE sinker together.
Place a 5 µm Versapor membrane disc filter on the housing base.
Seal the membrane with a membrane seal gasket and the upper housing.
Place the feeding tubing with PTFE sinker in the 750mL sample (see steps 1 and 2 from Before start section).
Turn on the pump (<15 psi). Release the thumb clamp of the feeding tube to allow the water sample to pass through. The water sample will be filtered and collected in the 1L sterile bottle. Turn off the vacuum pump after the water sample runs out.
Include a positive control for each batch of samples: transfer 37.5µL ZymoBIOMICS Microbial Community Standard and 100µL EBV, and 100µL HIV standard into 180mL sterile 1x PBS.
Include a negative control for each batch of samples: 180mL sterile 1x PBS.
Add 250mL of PEG-8000 solution to the pre-filtered 750mL water sample and 60mL of PEG-8000 solution in each control (the final solution of PEG-8000 is 10%). Mix well by shaking.
Store the sample at 4°C .
3. MICROORGANISM COLLECTION
Place a 300 ml MicroFunnel ST Filter funnel on a wire rack.
Connect the tubing from the Medi-Vac Canister to the base of a 300 ml MicroFunnel ST Filter Funnel.
Turn on the pump (<15 psi). Pour the PEG-8000 treated water sample into the filter funnel (Stir the water sample while pouring).
If possible, filter the entire 750mL water sample using one filter funnel (the filtrate will be collected in the Medi-Vac Canister as waste). When clogging happens, use a new funnel to filter the rest.
Disassemble the filter funnel. Carefully use an autoclaved forceps (or wipe with 70% ethanol and let dry) to move the GN-6 membrane on the supporting pad to a sterile 60 mm Petri dish.
If not processing the membrane immediately, evenly distribute 250µL DNA/RNA Shield Reagent on the membrane in the Petri dish, seal the Petri dish with parafilm and store at -20°C for short-term and -80°C for long-term until DNA/RNA extraction.
4. SAMPLE LYSIS
Pre-cool the Bullet Blender by adding dry ice into the cooling compartment and running the cooling program.
Clean the work surfaces with RNaseZap, then wipe the surfaces with 70% molecular biology grade ethanol to remove additional contaminants.
Transfer 0.1 mm zirconium oxide beads (2 spoons, Appendix 4, see Guidelines & Warnings tab) and four 3.5 mm UFO beads to Clear RINO brand 1.5 ml screw-cap microcentrifuge tubes.
Add 500µL of ATL-DX buffer and 135µL VXL buffer to the Clear RINO brand 1.5 ml screw-cap micro-centrifuge tubes containing 0.1 mm and 3.5 mm UFO beating beads.
Cut the membrane with a new razor blade into 2 halves.
Place a half of the filter membrane in a new Petri dish and store the unused half membrane in the Petri dish at -20°C for future use.
Use 70% ethanol to wipe forceps and surgical scissors. Use the forceps to fold the half membrane into a smaller size sector and directly cut the sector (into tiny pieces, smaller than 1 mm x 3 mm) into the 1.5 mL RINO tube with lysis buffer and beads.
Add 20µL Proteinase K from IndiMag kit and incubate the tube at 56°C in the heat block shaker set up at 1400rpm .
Load the sample/bead tubes in the Bullet Blender.
Set the speed at 12 and time at 3. Press Start.
Let the samples settle for 0h 1m 0s and then repeat step 32.
5. INSTRUMENT SET UP
Confirm 96 deep-well magnetic head and 96 well deep-well heat block are being used.
Ensure the program IndiMag_Pathogen_KF_Flex_4wash has been downloaded and loaded onto the KingFisher Flex instrument.
5.1 SET UP THE PROCESSING PLATES
Set up the Wash, Elution, and Tip Comb Plates outside the instrument according to the following table.
| A | B | C | D | E |
|---|---|---|---|---|
| Plate ID | Plate position | Plate type | Reagent | Volume per well |
| Tip comb | 7 | Place a 96 Deep-well Tip comb in a deep-well plate | ||
| Elution | 6 | Deep-Well | Nuclease-free water | 75 µL |
| Wash 4 | 5 | Deep-Well | 100 % ethanol | 750 µL |
| Wash 3 | 4 | Deep-Well | 80% ethanol | 750 µL |
| Wash 2 | 3 | Deep-Well | Buffer AW2 | 700 µL |
| Wash 1 | 2 | Deep-Well | Buffer AW1 | 700 µL |
| Sample | 1 | Sample Lysate | Lysate and lysis buffer | 985 µL |
5.2 EXTRACTION
Centrifuge the bead tubes with lysate from step 33 for 12000x g.
Transfer 425µL supernatant without any particle carryover to the wells of the Deep-well plate. This plate becomes the Sample Plate.
Add 540µL Buffer ACB, and 20µL MagAttract Suspension G to each sample in the sample plate. For multiple samples, make a master mix with 10% overage. Invert slowly to mix the master mix, avoid foaming (can be mixed on Hula mixer for 2 min). Add 560µL mixture to each sample.
Select the program IndiMag_Pathogen_KF_Flex_4wash on the instrument.
Start the run, then load the prepared plates into the positions when prompted by the instrument.
5.3 QUANTIFICATION AND STORAGE
After the running protocol is completed (~35 minutes), immediately remove the elution plate from the instrument and cover the plate or transfer the eluate to the final tube or plate of choice for final storage.
In a 0.6 mL microcentrifuge tube, use 3µL total nucleic acid for DNA and RNA concentration measurement using Qubit 4 Fluorometer following manufacturer instructions.
Proceed with sample testing following the REDI-NET SOP W-4 Water Testing or store at -20°C for less than 2 weeks.
6. INSTRUMENT SET UP
Confirm 12-tip magnetic head and 12 deep-well heat blocks are being used.
Ensure the program IndiMag_Pathogen_KF_Duo_4wash has been downloaded and loaded onto the KingFisher Duo Prime instrument.
6.1 SET UP SAMPLE PLATE AND ELUTION STRIP
Set up the Sample Plate according to the table below:
| A | B | C | D |
|---|---|---|---|
| Row ID | Plate Row | Reagent | Volume per well |
| Sample row | A | Lysate and lysis buffer | 985 µL |
| Wash 1 | B | Buffer AW1 | 700 µL |
| Wash 2 | C | Buffer AW2 | 700 µL |
| Wash 3 | D | 80 % ethanol | 750 µL |
| Wash 4 | E | 100 % ethanol | 750 µL |
| Tip Comb Wash 2 | F | 12-Tip comb | |
| G | Empty | ||
| H |
Set up the Elution Strip according to the table below:
| A | B | C | D |
|---|---|---|---|
| Row ID | Plate Row | Reagent | Volume per well |
| Elution | A | Nuclease-free water | 75 µL |
6.2 EXTRACTION
Centrifuge the bead tubes with lysate from step 33 for 12000x g.
Transfer 425µL supernatant without any particle carryover to the wells of the Deep-well plate. This plate becomes the Sample Plate.
Add 540µL Buffer ACB, and 25µL MagAttract Suspension G to each sample in the sample plate. For multiple samples, make a master mix with 10% overage. Invert slowly to mix the master mix, avoid foaming. Add 565µL mixture to each sample.
Select the program IndiMag_Pathogen_KF_Duo_4wash on the instrument.
Start the run, then load the prepared plates into position when prompted by the instrument.
Keep the door open while extraction is in process. The chamber of the KingFisher Duo Prime is small. Closing the door makes the ethanol vapor restrained inside the chamber and increases the ethanol contamination.
6.3 QUANTIFICATION AND STORAGE
After the running protocol is completed (~35 minutes), immediately remove the elution plate from the instrument and cover the plate or transfer the eluate to the final tube or plate of choice for final storage.
In a 0.6 mL microcentrifuge tube, use 3µL total nucleic acid for DNA and RNA concentration measurement using Qubit 4 Fluorometer following manufacturer instructions. (see Appendix 5 and Appendix 6).
Proceed with sample testing following the REDI-NET SOP W-4 Water Testing or store at -20°C for less than 2 weeks).
APPENDIX 3. PEG-8000 Preparation
Preparation of 40% PEG-8000 Solution
| A | B |
|---|---|
| PEG-8000 | 400 g |
| NaCl | 70 g |
| Add 1x PBS to final volume | 1 L |
The final concentrations: 40% PEG-8000 and 1.2M NaCl.
Add stir bar, PEG-8000 and NaCl into an empty 1L sterile bottle (plastic or glass, autoclavable).
Add sterile 1xPBS to final volume 1L.
Autoclave the whole bottle with lid at 121℃ for 30 min. After autoclave, place the hot solution on a stirring hot plate and stir the solution until it cools down to room temperature.
If autoclave is not available, stir the solution on a stirring hot plate until the crystals are fully dissolved and filter the solution through 0.45 µm Corning Disposable Vacuum Filter/Storage Systems.
Store the 40% PEG-8000 solution at 4℃.
APPENDIX 5. DNA and RNA Measurement using Qubit Fluorometer 4.0
DNA quantification:
According to the volume of sample used, add the 1xHS dsDNA Qubit Assay for a final volume of 200 µL (i.e., if using 3 µL of sample, add 197 µL of 1x HS dsDNA Qubit Assay.
RNA Quantification:
In a new microcentrifuge tube/falcon tube (depending on the number of samples processed), prepare a working solution of the Qubit HS RNA Assay:
In a new 0.6 ml tube, mix 197 µL of Qubit HS RNA Assay working solution and 3 µL of the sample. Incubate for 1 minute at room temperature before reading.
