Apr 07, 2023
QIAGEN DNeasy PowerSoil Kit
- QIAGEN1
- 1QIAGEN
Protocol Citation: QIAGEN 2023. QIAGEN DNeasy PowerSoil Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj8drpgk5/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 successful in several fish sedDNA studies
Created: December 21, 2022
Last Modified: April 18, 2023
Protocol Integer ID: 74339
Keywords: QIAGEN, DNeasy, PowerSoil, sedDNA, Sedimentary DNA, Fish
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
Materials
DNeasy PowerSoil Kit (50 preparations) already includes:
MB Spin Columns * 50
PowerBead Tubes * 50
Solution C1 6.6 mL
Solution C2 15 mL
Solution C3 15 mL
Solution C4 72 mL
Solution C5 30 mL
Solution C6 9 mL
Collection Tubes (2ml) 4 * 50
Quick Start Protocol
Additional equipments and reagents to be supplied by user:
Microcentrifuge (10.000 x g )
Pipettors (50 μl–500 μl)
Vortex-Genie 2 Vortex
Vortex Adapter for 24 (1.5-2.0 ml) tubes (cat. no. 13000-V1-24)
100% ethanol (for the QIAvac 24 Plus Manifold protocol only)
QIAvac 24 Plus Manifold
Safety warnings
Solution C5 contains ethanol. It is flammable.
Do not use bleach to clean the inside of the QIAvac® 24 Plus manifold.
Before start
Perform all centrifugation steps at room temperature (15–25°C).
If Solution C1 has precipitated, heat at 60°C until precipitate dissolves.
Sample preparation & cell lysis
Sample preparation & cell lysis
ADD 0.25 g of soil sample to the PowerBead Tube
VORTEX gently to mix
ADD 60 µL 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 00:10:00
10m
CENTRIFUGE tubes at 10.000 x g for 00:00:30
TRANSFER supernatant to a clean 2 mL Collection Tube
30s
Inhibitor removal
Inhibitor removal
25m 40s
25m 40s
ADD 250 µL of Solution C2
VORTEX for 00:00:05
INCUBATE at 2 °C to 8 °C for 00:05:00
5m 5s
CENTRIFUGE tubes at 10000 x g for 00:01:00
AVOIDING the pellet, transfer up to 600 µL of supernatant to a clean 2 mL Collection Tube
1m
ADD 200 µL of Solution C3
VORTEX briefly to mix
INCUBATE at 2 °C to 8 °C for 00:05:00
5m
CENTRIFUGE tubes at 10000 x g for 00:01:00
AVOIDING the pellet, transfer up to 750 µL of supernatant to a clean 2 mL Collection Tube
1m
Bind DNA
Bind DNA
3m 5s
3m 5s
SHAKE to mix Solution C4
ADD 1200 µL of Solution C4 to the supernatant
VORTEX for 00:00:05
5s
LOAD 675 µL onto an MB Spin Column
CENTRIFUGE at 10.000 x g for 00:01:00
DISCARD liquid flow-through
1m
REPEAT step 10 twice, until all of the sample has been processed
Wash spin column
Wash spin column
3m 5s
3m 5s
ADD 500 µL of Solution C5
CENTRIFUGE at 10.000 x g for 00:00:30
DISCARD liquid flow-through
30s
CENTRIFUGE again at 10.000 x g 00:01:00
CAREFULLY place the MB Spin Column into a clean 2 mL Collection Tube. Avoid splashing any residual Solution C5 onto the column
1m
Elute the DNA
Elute the DNA
3m 5s
3m 5s
ADD 100 µL of Solution C6 to the center of the white filter membrane
CENTRIFUGE at 10000 x g for 00:00:30
DISCARD the MB Spin Column
DNA is now ready for downstream applications
30s
Protocol references
Successful Publications:
Olajos, F., Bokma, F., Bartels, P., Myrstener, E., Rydberg, J., Öhlund, G., Bindler, R., Wang, X. R., Zale, R., & Englund, G. (2018). Estimating species colonization dates using DNA in lake sediment. Methods in Ecology and Evolution, 9(3), 535-543. https://doi.org/10.1111/2041-210X.12890
Nelson-Chorney, H. T., Davis, C. S., Poesch, M. S., Vinebrooke, R. D., Carli, C. M., & Taylor, M. K. (2019). Environmental DNA in lake sediment reveals biogeography of native genetic diversity. Frontiers in Ecology and the Environment, 17(6), 313-318. https://doi.org/10.1002/fee.2073.
Thomson-Laing, G., Parai, R., Kelly, L. T., Pochon, X., Newnham, R., Vandergoes, M. J., Howarth, J. D., & Wood, S. A. (2020). Development of droplet digital Polymerase Chain Reaction assays for the detection of long-finned (Anguilla dieffenbachii) and short-finned (Anguilla australis) eels in environmental samples. PeerJ, 9. https://doi.org/10.7717/peerj.12157
Naro-Maciel E, Ingala MR, Werner IE, Reid BN, Fitzgerald AM (2022) COI amplicon sequence data of environmental DNA collected from the Bronx River Estuary, New York City. Metabarcoding and Metagenomics 6: e80139. https://doi.org/10.3897/mbmg.6.80139