QIAGEN DNeasy PowerSoil Pro Kit
QIAGEN
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 Unpublished studies report poor DNA yields and low concentrations of fish sedDNA.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.
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).
Steps
Sample preparation & cell lysis
SPIN the PowerBead Pro Tube briefly to ensure that the beads have settled at the bottom
ADD up to 0.25g of soil sample to the PowerBead Pro Tube
ADD 800µL 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 0h 10m 0s
USING a PowerLyzer 24 Homogenizer, homogenize the soil at 2000rpm,0h 0m 0s for
PAUSE for 0h 0m 30s
HOMOGENIZE again at 2000rpm,0h 0m 0s for 0h 0m 30s
USING a TissueLyser II, place the PowerBead Pro Tube into the TissueLyser Adapter
FASTEN the adapter into the instrument and shake for 0h 5m 0s 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 0h 5m 0s at a speed of 25 Hz.
CENTRIFUGE the PowerBead Pro Tube at 15000x g,0h 0m 0s for 0h 1m 0s
TRANSFER supernatant to a clean 2 mL Microcentrifuge Tube (expect 500-600ul)
Inhibitor removal
ADD 200µL of Solution CD2
VORTEX for 0h 0m 5s
CENTRIFUGE tubes at 15000x g,0h 0m 0s for 0h 1m 0s
AVOIDING the pellet, transfer up to 700µL of supernatant to a clean 2 ml Microcentrifuge Tube
Bind DNA
ADD 600µL of Solution CD3
VORTEX for 0h 0m 5s
LOAD 650µL of the lysate onto an MB Spin Column
CENTRIFUGE at 15000x g,0h 0m 0s for 0h 1m 0s
DISCARD the liquid flow-through
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
ADD 500µL of Solution EA to the MB Spin Column
CENTRIFUGE at 15000x g,0h 0m 0s for 0h 1m 0s
DISCARD the flow-through and place the MB Spin Column into the same 2 mL Collection Tube
ADD 500µL of Solution C5 to the MB Spin Column
CENTRIFUGE at 15000x g,0h 0m 0s for 0h 1m 0s
DISCARD the flow-through and place the MB Spin Column into a NEW 2 mL Collection Tube
CENTRIFUGE at 16000x g,0h 0m 0s for 0h 2m 0s
CAREFULLY place the MB Spin Column into a new 1.5 ml Elution Tube
Elute the DNA
ADD between 50µL and 100µL of Solution C6 to the center of the white filter membrane
CENTRIFUGE at 15000x g for 0h 1m 0s
DISCARD the MB Spin Column
DNA is now ready for downstream applications
