Apr 06, 2023
QIAGEN DNeasy PowerSoil Pro Kit
Forked from QIAGEN® DNeasy® PowerSoil® Pro
- QIAGEN 1
- 1QIAGEN
Protocol Citation: QIAGEN 2023. QIAGEN DNeasy PowerSoil Pro Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx97mzg8j/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: Other
No published studies (as of April 2023) using this protocol to detect fish sedDNA
Unpublished studies on lake surface sediments report low detection of fish sedDNA
Protocol successful at detecting fish sedDNA collected in a stream during a fish migration
Created: December 21, 2022
Last Modified: April 18, 2023
Protocol Integer ID: 74332
Keywords: Qiagen, DNeasy, PowerSoil Pro, SedDNA, Sedimentary DNA, Fish, found sufficient fish seddna concentration, sufficient fish seddna concentration, low concentrations of fish seddna, detecting fish seddna, targeting migratory fish seddna, microbial genomic dna from environmental sample, genomic dna from environmental sample, microbial genomic dna from all soil type, isolating microbial genomic dna, qiagen dneasy powersoil pro kit for the isolation, qiagen dneasy powersoil pro kit, migratory fish seddna, fish seddna, microbial genomic dna, use with environmental sample, poor dna yield, quality dna, dneasy powersoil pro kit, difficult samples such as compost, genomic dna, containing high humic acid content, generation sequencing, high humic acid content, environmental sample, genomic, dna
Abstract
For the isolation of microbial genomic DNA from all soil types, including difficult samples such as compost, sediment, and manure.
The DNeasy PowerSoil Pro Kit comprises a novel and proprietary method for isolating microbial genomic DNA from environmental samples. The kit uses QIAGEN’s secondgeneration Inhibitor Removal Technology® (IRT) and is intended for use with environmental samples containing high humic acid content, including difficult soil types such as compost, sediment, and manure. Other more common soil and stool types have also been used successfully with this kit. Improved IRT combined with more efficient bead beating and lysis chemistry yields high-quality DNA that can be used immediately in downstream applications, including PCR, qPCR, and next-generation sequencing (16S and whole genome).
As of April, 2023 - no published studies successfully detecting fish sedDNA using only this kit (see Lakes ABPS protocol). Unpublished studies report poor DNA yields and low concentrations of fish sedDNA. Other studies targeting migratory fish sedDNA during a spawning run found sufficient fish sedDNA concentrations following this protocol.
Guidelines
Materials
DNeasy PowerSoil Pro Kit (50 preparations) already includes:
- PowerBead Pro Tubes * 50
- MB Spin Columns * 50
- Solution CD1 40 mL
- Solution CD2 15 mL
- Solution CD3 35 mL
- Solution EA 36 mL
- Solution C5 30 mL
- Solution C6 9 mL
- Microcentrifuge Tubes (2 mL) * 100
- Elution Tubes (1.5 mL) * 50
- Collection Tubes (2 mL) * 100
Equipment and Reagents to be Supplied by User
- Microcentrifuge (up to 16,000 x g)
- Pipettor (50-1000 ul)
- Vortex Genie
- Vortex Adapter for 24 (1.5-2 mL) tubes
Troubleshooting
Safety warnings
- Solution EA and Solution C5 are flammable
- DO NOT add bleach or acidic solutions directly to the sample preparation waste.
Before start
- Ensure that the PowerBead Pro Tubes rotate freely in the centrifuge without rubbing.
- If Solution CD3 has precipitated, heat at 60°C until precipitate dissolves.
- Perform all centrifugation steps at room temperature (15–25°C).
Sample preparation & cell lysis
SPIN the PowerBead Pro Tube briefly to ensure that the beads have settled at the bottom
ADD up to 0.25 g of soil sample to the PowerBead Pro Tube
ADD 800 µL of Solution CD1
VORTEX briefly to mix
HOMOGENIZE samples thoroughly using one of the following methods:
SECURE the PowerBead Pro Tube horizontally on a Vortex Adapter for 1.5–2 ml tubes
VORTEX at maximum speed for 00:10:00
10m
USING a PowerLyzer 24 Homogenizer, homogenize the soil at 2000 rpm for 00:00:00
PAUSE for 00:00:30
HOMOGENIZE again at 2000 rpm for 00:00:30
1m
USING a TissueLyser II, place the PowerBead Pro Tube into the TissueLyser Adapter
FASTEN the adapter into the instrument and shake for 00:05:00 at speed 25 Hz
REORIENT the adapter so that the side that was closest to the machine body is now furthest from it
SHAKE again for 00:05:00 at a speed of 25 Hz.
10m
CENTRIFUGE the PowerBead Pro Tube at 15000 x g for 00:01:00
TRANSFER supernatant to a clean 2 mL Microcentrifuge Tube (expect 500-600ul)
1m
Inhibitor removal
1m 5s
ADD 200 µL of Solution CD2
VORTEX for 00:00:05
5s
CENTRIFUGE tubes at 15000 x g for 00:01:00
AVOIDING the pellet, transfer up to 700 µL of supernatant to a clean 2 ml Microcentrifuge Tube
1m
Bind DNA
1m 5s
ADD 600 µL of Solution CD3
VORTEX for 00:00:05
5s
LOAD 650 µL of the lysate onto an MB Spin Column
CENTRIFUGE at 15000 x g for 00:01:00
DISCARD the liquid flow-through
1m
REPEAT step 7 to ensure that all of the lysate has passed through the MB Spin Column
CAREFULLY place the MB Spin Column into a clean 2 mL Collection Tube. Avoid splashing any flow-through onto the MB Spin Column
Wash spin column
4m
ADD 500 µL of Solution EA to the MB Spin Column
CENTRIFUGE at 15000 x g for 00:01:00
DISCARD the flow-through and place the MB Spin Column into the same 2 mL Collection Tube
1m
ADD 500 µL of Solution C5 to the MB Spin Column
CENTRIFUGE at 15000 x g for 00:01:00
DISCARD the flow-through and place the MB Spin Column into a NEW 2 mL Collection Tube
1m
CENTRIFUGE at 16000 x g for 00:02:00
CAREFULLY place the MB Spin Column into a new 1.5 ml Elution Tube
2m
Elute the DNA
1m
ADD between 50 µL and 100 µL of Solution C6 to the center of the white filter membrane
CENTRIFUGE at 15000 x g for 00:01:00
DISCARD the MB Spin Column
DNA is now ready for downstream applications
1m