Sep 01, 2023
Qiagen QIAmp PowerFecal Pro extraction kit
- Michael Dan Siemon1,
- Dilan Joseph1,
- Jessica Pardy1,
- Richard Gibson1,
- christopher.degroot1
- 1Western University
Protocol Citation: Michael Dan Siemon, Dilan Joseph, Jessica Pardy, Richard Gibson, christopher.degroot 2023. Qiagen QIAmp PowerFecal Pro extraction kit. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlko8owv5r/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
This protocol is used for wastwater RNA extraction for wastewater-based epidemiology at Western University
Created: August 30, 2023
Last Modified: September 01, 2023
Protocol Integer ID: 87125
Keywords: wastewater-based epidemiology, RNA extraction, Qiagen, ImPaKT
Abstract
This protocol is used by Western University to process wastewater samples for wastewater-based epidemiology. The protocol is modified from the protocol described in the RNeasy PowerFecal Pro Kit Handbook provided by Qiagen with the PowerFecal Pro kit.
Materials
This protocol requires the Qiagen QIAmp PowerFecal Pro extraction kit
Sample Preparation
Sample Preparation
Thoroughly mix wastewater sample then aliquot 40 mL into 50 mL Falcon tube. Centrifuge at 4500 x g for 120 min. Decant supernatant, assume 280 µl pellet.
Spin the PowerBead Pro Tube briefly to ensure that the beads have settled at the bottom. Add 100 μl of phenol−chloroform−isoamyl alcohol to the PowerBead Pro Tube.
Add 650 μl of Solution CD1 to the wastewater pellet and transfer solution to the PowerBead Pro Tube.
As a substitute for vortexing described in the kit protocol, bead-beating is used. Bead-beating is conducted 4x for 30s at 4 m/s.
Centrifuge the PowerBead Pro Tube at 15,000 x g for 1 min.
Transfer the supernatant to a clean 2 ml Microcentrifuge Tube. Add 200 μl of Solution CD2 and vortex for 5 s.
Centrifuge at 15,000 x g for 1 min. Avoiding the pellet, transfer up to 650 μl of supernatant to a clean 2 ml Microcentrifuge Tube.
Add 650 μl of Solution EA. Vortex briefly.
Load 650 μl supernatant-EA mix into an MB RNA Spin Column and centrifuge at 15,000
x g for 1 min. Discard the flow-through and repeat until all of the solution has been passed through the Spin Column.
Add 650 μl Solution EA to the Spin Column and centrifuge at 15,000 x g for 1 min.
Mix 45 μl DNase Digestion Solution and 5 μl DNase I stock enzyme. Place the MB RNA Spin Column into a clean 2 ml Collection Tube and add the DNase Digestion Solution to the center of the filter. Incubate at room temperature for 15 minutes.
Add 650 μl Solution EA to the Spin Column and centrifuge at 15,000 x g for 1 min.
Discard the flow-through. Add 500 μl Solution C5. Centrifuge at 15,000 x g for 1 min.
Replace the collection tube and dry the Spin Column by centrifuging at 20,000 x g for 1 min.
Elute the RNA by placing the spin column in a 1.5 ml collection tube. Add 100 μl of RNase-free water and centrifuge at 15,000 x g for 1 min.
Protocol references
This protocol is modified from the protocol described in RNeasy PowerFecal Pro Kit Handbook:
RNeasy PowerFecal Pro Kit Handbook - Qiagen (2022). Retrieved August 31, 2023 from: