Nov 13, 2024
Automated Extraction of Concentrated Wastewater Samples using Promega HT Environmental TNA Kit
- 1University of Wisconsin Madison;
- 2Wisconsin State Laboratory of Hygiene
- Coronavirus Method Development Community
- APHL Wastewater Surveillance Community of Practice
Protocol Citation: Dagmara Antkiewicz, Adelaide Roguet, Ashlie McCunn 2024. Automated Extraction of Concentrated Wastewater Samples using Promega HT Environmental TNA Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzx4wxgx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 05, 2024
Last Modified: November 13, 2024
Protocol Integer ID: 96174
Funders Acknowledgement:
WI DHS ELC
Grant ID: 435100-A23-ELCProje-00
Abstract
This protocol describes how to extract total nucleic acids (TNA) from viruses and bacteria concentrated from environmental samples such as wastewater, drinking water, surface and recreational waters. The resulting TNA is suitable for downstream molecular analyses. This method uses the KingFisherTM Flex automated system with the DNA/RNA extraction kit called Promega Maxwell‱ HT Environmental TNA Kit, AX9190.
Guidelines
For laboratories performing a concentration method prior to extraction, it is recommended to start setting up this automated extraction protocol 15 to 30 minutes before the concentration method is finished. Our laboratory's concentration protocol can be found here: dx.doi.org/10.17504/protocols.io.261gedz7dv47/v1.
Materials
Equipment and Supplies:
Refrigerator storage (4°C)
Pipettes and filtered pipette tips
Conical centrifuge tubes
KingFisherTM Deep Well 96 Plate, barcoded (catalog# 95040450B)
KingFisherTM 96 Tip Comb for DW Magnets (catalog# 97002534)
Thermo Scientific KingFisherTM Flex (catalog# N07669)
Round-bottom sterile 96-well plates (e.g., Cell Treat catalog# 229590)
Plate sealing film (e.g., Axygen‱ catalog# PCR-AS-600)
Absorbent sheets
Kimtech science Kimwipes
Reagents and Standards:
50% Ethanol solution (using 200 Proof absolute non-denatured ethanol and sterile molecular-grade water)
100% Isopropanol
Custom Promega Maxwell‱ HT Environmental TNA Kit, catalog# AX9190 [Includes: Alkaline Protease solution, Cell Lysis Buffer (CLD), Resin, Wash Buffer, and 25mM Tris-HCL (pH 8.0). The HT Environmental TNA kit can be purchased here.]
10% Bleach solution
ELIMINaseTM (or another reagent that hydrolyzes DNA and RNA)
70% Ethanol solution (e.g., 200 Proof absolute non-denatured ethanol and sterile molecular-grade water)
Software:
KingFisherTM Promega Maxwell HT RNA Wastewater V1: 
Promega_Maxwell_HT_RNA_Wastewater_V1.bdz3KB
Promega_Maxwell_HT_RNA_Wastewater_V1.bdz3KB Protocol:
Ceres Nanosciences, Inc. has published recommendations for use of the Promega Maxwell‱ HT Environmental TNA Kit in this protocol (starting at Step 2 of the procedure). Our laboratory follows Ceres's extraction recommendations with a few exceptions: using the Thermo Scientific KingFisherTM Flex, adding isopropanol after the sample in the Lysis/Bind (Bead Binding) plate, eluting in 200μL of Tris-HCl, and using the Promega_Maxwell_HT_RNA_Wastewater_V1.bdz software program.
Before start
When using the benchtop, clean workspace area with 10% bleach, followed by ELIMINaseTM, and then 70% ethanol before and after use.
Obtain Concentrated Samples
Obtain Concentrated Samples
5m
5m
Obtain wastewater samples that have undergone a concentration method.
If concentrated samples are in storage, remove from them from the freezer and let them thaw on the benchtop.
Reagent Preparation
Reagent Preparation
8m
8m
Prepare single use volumes of clean reagents (that are not provided in the Promega kit) each day for a batch up to 40 samples:
Pipette 30 mL of molecular-grade 100% isopropanol into a 50mL conical centrifuge tube.
Pipette 25 mL sterile molecular-grade water followed by 25 mL of 200 proof ethanol into 50mL conical centrifuge tube. Invert the conical gently to mix the 50% ethanol solution.
Extraction Plates
Extraction Plates
30m
30m
Label five KingFisher Deep Well 96 Plates "Wash 1", "Wash 2", "Ethanol" (EtOH), "Elution", and "Lysis/Bind".
Note
Plating the samples in a checkerboard pattern can help prevent well contamination if foaming occurs during the mixing steps while on the KingFisherTM Flex instrument.
For the Ethanol plate: add 450 µL of 50% ethanol solution to each of the sample wells as indicated on your plate map.
For the Wash plates: add 100 µL of 50% ethanol solution to each of the sample wells in both the Wash 1 and Wash 2 plates.
Then, add 900 µL of Wash Buffer to all sample wells in both Wash 1 and Wash 2 plates.
For the Elution plate: add 200 µL of 25 mM Tris-HCL to the sample wells according to your plate map.
Assemble the Lysis/Bind plate in the following order:
Add 50 µL of Alkaline Protease solution to the sample wells.
Vortex the Resin thoroughly (15-20 seconds). Then, add 35 µL of Resin to each sample well.
Transfer 300 µL of concentrated wastewater sample into its corresponding well in the Lysis/Bind plate according to the plate map. Repeat for all samples in the batch.
For the extraction blank, add 300 µL of Cell Lysis Buffer (CLD) in place of concentrated wastewater sample.
Add 400 µL of 100% isopropanol to each sample well.
Note
The isopropanol prevents foaming of the solution. If isopropanol is added before the sample, it can impact the binding of nucleic acid particles with the Resin.
Note
The volumes of the reagents in the Lysis/Bind plate can be adjusted to perform extraction following various concentration procedures. The reagent variations can be found in the table below:
Insert a KingFisher 96 Tip Comb for DW Magnet into a sixth KingFisher Deep Well 96 Plate.
Loading KingFisher Flex
Loading KingFisher Flex
3m
3m
Go to the menu screen on the KingFisher Flex, and select the program titled "Promega Maxwell HT RNA Wastewater V1".
Slide the door of the KingFisher Flex open, and press start to proceed.
Follow the instrument prompts to load the plates into the plate trays. Ensure all 96-well plates are loaded with A1 corner in the upper right corner of the instrument's plate tray.
Close the door and start the program. The program will run for 1 hour and 15 minutes.
1h 15m
Unloading KingFisher Flex
Unloading KingFisher Flex
1m
1m
When the Promega Maxwell HT RNA Wastewater V1 program is finished, remove all plates from the Kingfisher Flex as instructed by the instrument prompts.
Keep the Elution plate, and set it aside to use in the next step.
Discard the Wash 1, Wash 2, Ethanol, Lysis/Bind, and Tip plates according to your biosafety protocol.
Final Plating of Total Nucleic Material
Final Plating of Total Nucleic Material
30m
30m
Transfer the entire volume (around 200 µL ) of extracted nucleic material from the Elution plate to a round-bottom sterile 96-well plate (pictured below). Repeat this step for each sample.
Cover the sterile 96-well plate with an aluminum plate sealing film.
Store the 96-well plate at 4 °C if the sample is going to be analyzed the same day.
If the nucleic acid material is not being analyzed that same day of extraction, store in a freezer at -70 °C or below for up to 2 years.