Sep 09, 2021
Version 4
Total nucleic acid extraction - Maxwell(R) HT Environmental TNA Kit, custom (Promega) V.4
- 1UWM
- mclellan lab
Protocol Citation: Adélaïde Roguet 2021. Total nucleic acid extraction - Maxwell(R) HT Environmental TNA Kit, custom (Promega). protocols.io https://dx.doi.org/10.17504/protocols.io.bujgnujwVersion created by McLellan Lab
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: In development
We are still developing and optimizing this protocol
Created: April 27, 2021
Last Modified: September 09, 2021
Protocol Integer ID: 49480
Keywords: wastewater, SARS-CoV-2, total nucleic extraction, RNA, DNA, extraction, purification, Promega, KingFisher
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Abstract
Total nucleic acid extraction from wastewater using Maxwell(R) HT Environmental TNA Kit, custom (Promega)
Guidelines
When work is completed, remove equipment and supplies from the cabinet. Wipe the work area with 10% bleach, let stand for 10 min, rinse with water, then with 70% ethanol, and finally with RNAase AWAY.
Materials
MATERIALS
•Ethanol USP/ACS or molecular biology grade (100%)
•Molecular biology grade water
•Isopropanol molecular biology grade (100%)
•6x KingFisher 96-well plates (Cat. no.: 95040460)
•1x KingFisher 96-tip comb well plate(Cat. no.: 97002534)
•Screw cap microcentrifuge tubes
Equipment
Mini-Beadbeater-16
NAME
high-energy cell disrupter
TYPE
BioSpec
BRAND
607
SKU
LINK
1 speed
SPECIFICATIONS
Equipment
Kingfisher Flex
NAME
Automated Extraction System
TYPE
ThermoFisher
BRAND
5400630
SKU
Promega_Maxwell_HT_RNA_Wastewater_V1.bdz Before start
1. Clean the working area and all equipment: wipe down with 10% bleach and let dry. Wipe down with 70% ethanol and let dry. Then, wipe down using RNase AWAY and let dry.
2. Prepare the 50% ethanol solution (it must be fresh!)
3. Prepare the 6 purification plates:
- Wash 1 plate: Add 100 μl of 50% ethanol and 900 μl of wash buffer (WBA) to each well required for purification.
- Wash 2 plate (same as plate Wash 1): Add 100 μl of 50% ethanol and 900 μl of wash buffer (WBA) to each well required for purification.
- Ethanol Wash plate: Add 450 μl of 50% ethanol to each well required for purification.
- Elution plate: Add 100 μl of 25 mM Tris-HCl (pH 8.0) to each well required for purification.
- Tip plate: Place KingFisher 96-tip comb into an empty KingFisher 96-well plate. While opening the 96-tip comb plate, pay attention to not touch the tips.
- Lysis and Bind plate: Add 35 μL of Resin to each well required for purification (vortex the bottle at max speed before use). Add 50 μl of Alkaline Protease Solution custom (APA) to each well required for purification. Add 250 μl of cell lysis solution (CLD) to each well required for purification. Add 400 μl of Isopropanol (100%) to each well required for purification.
Total nucleic acid extraction
Total nucleic acid extraction
2h
2h
For HA filter extraction, let the sample thaw on ice and go to step 2.
For BCoV/BRSV extraction (in duplicate), add 5 µL of BCoV/BRSV solution to the 2-mL tube containing 250 µL CTAB. Vortex for 15 seconds (speed 7 out of 10) and flash freeze the tube. Go to step 4.
For Direct extraction, add 150 µL of wastewater to the 2-mL tube containing 100 µL CTAB. Vortex for 15 seconds (speed 7 out of 10) and flash freeze the tube. Go to step 4.
5m
For HA filter extraction, place the 2-mL tubes in the bead beater.
Equipment
Mini-Beadbeater-16
NAME
high-energy cell disrupter
TYPE
BioSpec
BRAND
607
SKU
LINK
1 speed
SPECIFICATIONS
Bead beat for 00:02:30
Safety information
Start the bead beating when the beads start to be loose in the tubes.
2m 30s
Cooldown the samples on ice for 00:05:00 .
5m
Repeat Steps 9.1 and 9.2 once go to step #2.1 .
7m 30s
Centrifuge at maximum speed for 1 min at room temperature. 150000 rpm, Room temperature, 00:01:00
1m
For HA filter extraction, transfer 125-250 µL of supernatant to the Lysis/Bind plate.
For BCoV/BRSV/Direct extraction, transfer all supernatant to the Lysis/Bind plate.
Note
The default volume transferred is 250 µL. However, for WWTPs with "dirty" influents, we only transfer 125 µL.
10m
Start the protocol Promega_Maxwell_HT_RNA_Wastewater_V1.bdz on the KingFisher Flex 01:14:00
Equipment
Kingfisher Flex
NAME
Automated Extraction System
TYPE
ThermoFisher
BRAND
5400630
SKU
1h 14m
Transfer the purified sample from the Elution plate to the microcentrifuge tubes.
Note
The DNA/RNA is now ready for downstream applications. RNA extract may be stored in RNase-free water at -80ºC for 1 year.
10m
RT-ddPCR
RT-ddPCR