QIAGEN DNeasy PowerMax Soil Kit
QIAGEN
Abstract
For the isolation of microbial DNA from large quantities of soil - great for samples with low microbial load
The DNeasy PowerMax Soil Kit comprises a novel and proprietary method for isolating genomic DNA from environmental samples using Inhibitor Removal Technology® (IRT). With this kit, it is possible to process samples that have proven difficult in the past due to high levels of humic-like substances. The isolated DNA has a high level of purity, which allows for successful PCR amplification from samples. Total DNA isolated from various soil types has been successfully amplified using PCR with primers specific for bacteria (Bacillus subtilis, Bacillus anthracis), fungi (yeast, mold), and actinomycetes (Streptomyces).
Using the DNeasy PowerMax Soil Kit, environmental samples are added to a bead-beating tube with a kit-supplied proprietary buffer for rapid and thorough homogenization. Cell lysis and DNA exposure occur by mechanical and chemical methods. Extracted genomic DNA is captured on a silica membrane in a spin column format. The DNA is washed and eluted from the membrane and is ready for PCR and other downstream applications.
Protocol successfully used by Sales et al., 2021 to characterize fish species diversity and richness in water and sediment samples
Steps
Sample preparation & cell lysis
ADD 15mL of PowerBead Solution to a PowerMax Bead Pro Tube
ADD up to 10g of soil sample to the PowerMax Bead Pro Tube containing PowerBead Solution
VORTEX vigorously for 0h 1m 0s
ADD 1.2mL of Solution C1 to the PowerMax Bead Pro Tube
VORTEX vigorously for 0h 0m 30s
PLACE PowerMax Bead Pro Tube on a vortex adapter
VORTEX for 0h 10m 0s at the highest speed
ALTERNATIVELY , place the tube in a shaking water bath set at 65°C and shake at maximum speed for 0h 30m 0s
CENTRIFUGE at 2500x g,0h 0m 0s for 0h 3m 0s at Room temperature
TRANSFER supernatant to a clean collection tube
Inhibitor removal
ADD 5mL of Solution C2
INVERT twice to mix
INCUBATE at 2°C to 8°C for 0h 10m 0s
CENTRIFUGE at 2500x g,0h 0m 0s for 0h 4m 0s at Room temperature
AVOIDING the pellet, transfer the supernatant to a clean collection tube
ADD 4mL of Solution C3
INVERT twice to mix
INCUBATE at 2°C to 8°C for 0h 10m 0s
CENTRIFUGE at 2500x g,0h 0m 0s for 0h 4m 0s at Room temperature
AVOIDING the pellet, transfer the supernatant to a clean collection tube
Bind DNA
SHAKE to mix Solution C4
ADD 30mL of Solution C4 to supernatant
INVERT twice to mix
FILL an MB Maxi Spin Column with the solution from step 9
CENTRIFUGE at 2500x g for 0h 2m 0s at Room temperature
DISCARD the liquid flow-through
ADD a second volume of supernatant to the same MB Maxi Spin Column
CENTRIFUGE at 2500x g for 0h 2m 0s at Room temperature
DISCARD the liquid flow-through
REPEAT until the entire volume has been processed. This will take up to 4 total spins
Wash spin column
ADD 10mL of Solution C5
CENTRIFUGE at 2500x g,0h 0m 0s for 0h 3m 0s at Room temperature
DISCARD the liquid flow-through
CENTRIFUGE again at 2500x g for 0h 5m 0s at Room temperature to remove residual Solution C5
CAREFULLY place the MB Maxi Spin Column in a new collection tube. Avoid splashing any residual SolutioN C5 onto the column
Elute the DNA
ADD 5mL of sterile Solution C6 to the center of MB Maxi Spin Column membrane
CENTRIFUGE at 2500x g,0h 0m 0s for 0h 3m 0s at Room temperature
DISCARD the MB Maxi Spin Column
DNA is now ready for downstream applications
