Aug 21, 2023
Soil DNA Extraction Modified Protocol for Dryland Agroecosystems
- McKenzie L. Stock1,
- Paul Gabriel1,
- Jennifer J. Randall1,
- Nicole Pietrasiak2
- 1New Mexico State University;
- 2University of Nevada Las Vegas
Protocol Citation: McKenzie L. Stock, Paul Gabriel, Jennifer J. Randall, Nicole Pietrasiak 2023. Soil DNA Extraction Modified Protocol for Dryland Agroecosystems. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly7domlx9/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
We use this protocol and it's working
Created: September 19, 2022
Last Modified: August 21, 2023
Protocol Integer ID: 70247
Funders Acknowledgement:
New Mexico Department of Agriculture Specialty Crop Block Grant
Grant ID: AM#21SCBPNM1027
Abstract
The Qiagen DNEasy PowerLyzer PowerSoil Kit is widely used in DNA based sample processing and extraction procedures. However, dryland soils are typically high in salts and secondary metabolites, which can cause interferences with A 260/230 quality of extracted DNA. We modified the Qiagen manufacturer protocol to account for high soil salinity by including additional washing steps and extra centrifugation time. Quality DNA was extracted from agricultural soils using this protocol which passed the quality checks for next generation amplicon sequencing.
Sample Prep
Sample Prep
Soil samples should be stored cold, at least -20 C (ideally at -80 C).
Homogenization
Homogenization
3m
3m
Fill a 12 ml homogenization vial approximately 2/3 full with soil (Spex SamplePrep 6133PC-T). Include three 6.35 mm diameter chrome steel beads (BioSpec Products Cat. No. 11079635c).
Homogenize at 4000 rpm for 00:00:10 using the SPEX SamplePrep 1200 Genolyte homogenizer with the single-vial attachment for 12mL vials. Re-homogenize as needed at 4000 rpm for 00:00:10 to avoid the soil from thawing and sticking together.
20s
DNA Extraction
DNA Extraction
17m 10s
17m 10s
This protocol is modified from the Qiagen manufacturer protocol for the DNeasy PowerLyzer PowerSoil Kit.
Equipment
DNeasy PowerLyzer PowerSoil Kit
NAME
DNA Extraction Kit
TYPE
Qiagen
BRAND
12855-50
SKU
LINK
.
Add approximately 80 µL of soil sample by volume to the PowerBead Tube provided.
Add 750 µL of PowerBead Solution to the PowerBead Tube.
Add 60 µL of Solution C1 and invert several times or vortex briefly.
Bead beating: Spex 1200 Genolyte homogenizer: Place the PowerLyzer Glass Bead Tubes into the tube holder attachment for the homogenizer (holds 6). The PowerBead Tubes must be balanced on the tube holder. Run the samples at a time and RPM suitable for your soil type. For our samples, 3000 rpm for 00:00:35 .
35s
Centrifuge Bead Tubes at 10,000 x rcf for 00:03:00 . Note: We increased the centrifugation time to ensure that no sediment/soil is left in suspension, improving DNA quality downstream.
3m
Transfer the supernatant to a clean 2 ml collection tube. Note: Expect 400-500 µL . Supernatant may still contain some soil particles.
Add 250 µL of Solution C2 and vortex for 5 sec. Incubate at 2-8°C for 5 min.
Centrifuge the tubes at 10,000 x rcf for 1 min. Avoiding the pellet, transfer up to 600 µL of supernatant to a clean 2 ml collection tube.
Add 200 µL of Solution C3 and vortex briefly. Incubate at 2-8°C for 00:05:00 .
5m
Centrifuge the tubes at 10,000 x rcf for 00:03:00 . Avoiding the pellet, transfer up to 750 µL of supernatant to a clean 2 ml collection tube. Note: We increased the centrifugation time to ensure that no sediment/soil is left in suspension, improving DNA quality downstream.
3m
Add 1200 µL of Solution C4 to the supernatant and vortex for 00:00:05 .
5s
Load 675 µL of the supernatant onto a MB Spin Column and centrifuge at 10,000 x rcf for 00:01:00 . Discard the flow through and add an additional 675 mL of supernatant.
1m
Centrifuge at 10,000 x rcf for 00:01:00 . Load the remaining supernatant onto the MB Spin Column and centrifuge at 10,000 rcf for 00:01:00 . Note: A total of three loads for each sample processed are required.
2m
Add 500 μl of Solution C5 and centrifuge at 10,000 x rcf for 00:00:30 . Discard the flow through. Repeat this step once more: add 500 µL of Solution C5 and centrifuge at 10,000 x rcf for 00:00:30 . Discard the flow through. Note: We repeated this washing step to minimize salt contamination, which helps improve DNA A260/230 values.
1m
Centrifuge again at 10,000 x rcf for 00:01:00 .
1m
Carefully place the spin filter in a clean 2 ml collection tube. Avoid splashing any Solution C5 onto the MB Spin Column.
Add 50 µL of Solution C6 to the center of the white filter membrane. Note: We used the minimum recommended amount of Solution C6 to yield greatest DNA concentrations.
Centrifuge at 10,000 x rcf for 00:00:30 . Discard the MB Spin Column.
30s
The DNA is now ready for downstream applications.