Apr 07, 2023
Version 1
QIAGEN DNeasy PowerMax Soil Kit V.1
- QIAGEN1
- 1QIAGEN
Protocol Citation: QIAGEN 2023. QIAGEN DNeasy PowerMax Soil Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr4ke3gmk/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: Working
Protocol used successfully by Sales et al., 2021 to detect fish sedDNA
Created: December 21, 2022
Last Modified: April 18, 2023
Protocol Integer ID: 74334
Keywords: QIAGEN, DNeasy, PowerMax, SedDNA, Sedimentary DNA
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
Materials
DNeasy PowerMax Soil Kit (10 preparations) already includes:
MB Maxi Spin Columns * 10
PowerMax Bead Pro Tubes * 10
PowerBead Solution 20 mL
Solution C1 6.6 mL * 2
Solution C2 28 mL * 2
Solution C3. 44 mL
Solution C4 330 mL
Solution C5 30 mL * 4
Solution C6 66 mL
Collection Tubes (50ml) 5 * 8
Quick Start Protocol * 1
Additional equipments and reagents to be supplied by user:
Centrifuge capable of spinning 50 ml tubes at 2500 x g using swing-out rotor
Pipettes (1ml and 10 ml)
Vortex-Genie 2 Vortex
Vortex Adapter for 2 (50 ml) tubes (cat. no. 13000-V1-50)
Safety warnings
Solution C5 contains ethanol and is flammable.
DO NOT add bleach or acidic solutions directly to the sample preparation waste.
Sample preparation & cell lysis
Sample preparation & cell lysis
1h 27m 30s
1h 27m 30s
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 00:01:00
Note
Refer to manufacturer's Troubleshooting Guide before deciding on the amount of soil to process. However, higher volumes of sediment (10 g) have shown better detection rates for fish sedDNA.
1m
ADD 1.2 mL of Solution C1 to the PowerMax Bead Pro Tube
VORTEX vigorously for 00:00:30
30s
PLACE PowerMax Bead Pro Tube on a vortex adapter
VORTEX for 00:10:00 at the highest speed
ALTERNATIVELY, place the tube in a shaking water bath set at 65 °C and shake at maximum speed for 00:30:00
40m
CENTRIFUGE at 2500 x g for 00:03:00 at Room temperature
TRANSFER supernatant to a clean collection tube
3m
Inhibitor removal
Inhibitor removal
1h 27m 30s
1h 27m 30s
ADD 5 mL of Solution C2
INVERT twice to mix
INCUBATE at 2 °C to 8 °C for 00:10:00
10m
CENTRIFUGE at 2500 x g for 00:04:00 at Room temperature
AVOIDING the pellet, transfer the supernatant to a clean collection tube
4m
ADD 4 mL of Solution C3
INVERT twice to mix
INCUBATE at 2 °C to 8 °C for 00:10:00
10m
CENTRIFUGE at 2500 x g for 00:04:00 at Room temperature
AVOIDING the pellet, transfer the supernatant to a clean collection tube
4m
Bind DNA
Bind DNA
1h 27m 30s
1h 27m 30s
SHAKE to mix Solution C4
ADD 30 mL of Solution C4 to supernatant
INVERT twice to mix
FILL an MB Maxi Spin Column with the solution from step 9
CENTRIFUGE at 2500 x g for 00:02:00 at Room temperature
DISCARD the liquid flow-through
2m
ADD a second volume of supernatant to the same MB Maxi Spin Column
CENTRIFUGE at 2500 x g for 00:02:00 at Room temperature
DISCARD the liquid flow-through
REPEAT until the entire volume has been processed. This will take up to 4 total spins
2m
Wash spin column
Wash spin column
1h 27m 30s
1h 27m 30s
ADD 10 mL of Solution C5
CENTRIFUGE at 2500 x g for 00:03:00 at Room temperature
DISCARD the liquid flow-through
3m
CENTRIFUGE again at 2500 x g for 00:05:00 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
5m
Elute the DNA
Elute the DNA
1h 27m 30s
1h 27m 30s
ADD 5 mL of sterile Solution C6 to the center of MB Maxi Spin Column membrane
CENTRIFUGE at 2500 x g for 00:03:00 at Room temperature
DISCARD the MB Maxi Spin Column
DNA is now ready for downstream applications
3m
Protocol references
Successful fish sedDNA publications using this protocol:
Sales, N. G., Wangensteen, O. S., Carvalho, D. C., Deiner, K., Præbel, K., Coscia, I., McDevitt, A. D., & Mariani, S. (2021). Space-time dynamics in monitoring neotropical fish communities using eDNA metabarcoding. Science of The Total Environment, 754, 142096. https://doi.org/10.1016/j.scitotenv.2020.142096