TITLE: QIAGEN DNeasy PowerMax Soil Kit
AUTHORS: QIAGEN

[DESCRIPTION]
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]
SECTION: Sample preparation &amp; cell lysis
1. ADD 15 mL of PowerBead Solution to a PowerMax Bead Pro Tube

ADD up to 10 g of soil sample to the PowerMax Bead Pro Tube containing PowerBead Solution

VORTEX vigorously for 1 min 
 

SECTION: Sample preparation &amp; cell lysis
2. ADD 1.2 mL of Solution C1 to the PowerMax Bead Pro Tube

VORTEX vigorously for 30 s
SECTION: Sample preparation &amp; cell lysis
3. PLACE PowerMax Bead Pro Tube on a vortex adapter

VORTEX for 10 min at the highest speed

ALTERNATIVELY, place the tube in a shaking water bath set at 65 °C and shake at maximum speed for 30 min
SECTION: Sample preparation &amp; cell lysis
4. CENTRIFUGE at 2500 x g for 3 min at Room temperature

TRANSFER supernatant to a clean collection tube
SECTION: Inhibitor removal
5. ADD 5 mL of Solution C2

INVERT twice to mix

INCUBATE at 2 °C to 8 °C for 10 min
SECTION: Inhibitor removal
6. CENTRIFUGE at 2500 x g for 4 min at Room temperature

AVOIDING the pellet, transfer the supernatant to a clean collection tube
SECTION: Inhibitor removal
7. ADD 4 mL of Solution C3

INVERT twice to mix

INCUBATE at 2 °C to 8 °C for 10 min
SECTION: Inhibitor removal
8. CENTRIFUGE at 2500 x g for 4 min at Room temperature

AVOIDING the pellet, transfer the supernatant to a clean collection tube
SECTION: Bind DNA
9. SHAKE to mix Solution C4

ADD 30 mL of Solution C4 to supernatant

INVERT twice to mix
SECTION: Bind DNA
10. FILL an MB Maxi Spin Column with the solution from step 9

CENTRIFUGE at 2500 x g for 2 min at Room temperature 

DISCARD the liquid flow-through
SECTION: Wash spin column
12. ADD 10 mL of Solution C5

CENTRIFUGE at 2500 x g for 3 min at Room temperature

DISCARD the liquid flow-through
SECTION: Elute the DNA
14. ADD 5 mL of sterile Solution C6 to the center of MB Maxi Spin Column membrane

CENTRIFUGE at 2500 x g for 3 min at Room temperature

DISCARD the MB Maxi Spin Column

DNA is now ready for downstream applications
SECTION: Bind DNA
11. ADD a second volume of supernatant to the same MB Maxi Spin Column

CENTRIFUGE at 2500 x g for 2 min at Room temperature 

DISCARD the liquid flow-through

REPEAT until the entire volume has been processed. This will take up to 4 total spins

SECTION: Wash spin column
13. CENTRIFUGE again at 2500 x g for 5 min 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