QIAGEN DNeasy PowerSoil Kit
QIAGEN
Abstract
For the isolation of microbial genomic DNA from all soil types
The DNeasy PowerSoil Kit comprises a novel and proprietary method for isolating genomic DNA from environmental samples using patented Inhibitor Removal Technology® (IRT). This kit 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 types have also been used successfully with this kit. The isolated DNA has a high level of purity, which allows for more successful PCR amplification of organisms from the sample. PCR analysis has been performed to detect a variety of organisms including bacteria (e.g., Bacillus subtilis, Bacillus anthracis), fungi (e.g., yeasts, molds), algae and actinomycetes (e.g., Streptomyces).
Protocol used successfully to detect target fish species sedDNA by Olajos et al., 2018; Nelson-Chorney et al., 2019; Thomson-Laing et al., 2020; Shiragaki et al., 2021; and Naro-Maciel et al., 2022
Before start
Perform all centrifugation steps at room temperature (15–25°C).
If Solution C1 has precipitated, heat at 60°C until precipitate dissolves.
Steps
Sample preparation & cell lysis
ADD 0.25g of soil sample to the PowerBead Tube
VORTEX gently to mix
ADD 60µL of Solution C1
INVERT several times or vortex briefly
SECURE PowerBead Tubes horizontally using a Vortex Adapter for 24 (1.5–2.0 ml) tubes
VORTEX at maximum speed for 0h 10m 0s
CENTRIFUGE tubes at 10.000x g,0h 0m 0s for 0h 0m 30s
TRANSFER supernatant to a clean 2 mL Collection Tube
Inhibitor removal
ADD 250µL of Solution C2
VORTEX for 0h 0m 5s
INCUBATE at 2°C to 8°C for 0h 5m 0s
CENTRIFUGE tubes at 10000x g,0h 0m 0s for 0h 1m 0s
AVOIDING the pellet, transfer up to 600µL of supernatant to a clean 2 mL Collection Tube
ADD 200µL of Solution C3
VORTEX briefly to mix
INCUBATE at 2°C to 8°C for 0h 5m 0s
CENTRIFUGE tubes at 10000x g for 0h 1m 0s
AVOIDING the pellet, transfer up to 750µL of supernatant to a clean 2 mL Collection Tube
Bind DNA
SHAKE to mix Solution C4
ADD 1200µL of Solution C4 to the supernatant
VORTEX for 0h 0m 5s
LOAD 675µL onto an MB Spin Column
CENTRIFUGE at 10.000x g,0h 0m 0s for 0h 1m 0s
DISCARD liquid flow-through
REPEAT step 10 twice, until all of the sample has been processed
Wash spin column
ADD 500µL of Solution C5
CENTRIFUGE at 10.000x g,0h 0m 0s for 0h 0m 30s
DISCARD liquid flow-through
CENTRIFUGE again at 10.000x g,0h 0m 0s 0h 1m 0s
CAREFULLY place the MB Spin Column into a clean 2 mL Collection Tube. Avoid splashing any residual Solution C5 onto the column
Elute the DNA
ADD 100µL of Solution C6 to the center of the white filter membrane
CENTRIFUGE at 10000x g for 0h 0m 30s
DISCARD the MB Spin Column
DNA is now ready for downstream applications
