QIAGEN® DNeasy® PowerSoil® Pro

Hsin-Mao Wu

Published: 2022-09-10 DOI: 10.17504/protocols.io.bp2l69411lqe/v1

Abstract

For the isolation of microbial genomic DNA from all soil types, including difficult samples such as compost, sediment, and manure.

Before start

Ensure that the PowerBead Pro Tubes rotate freely in the centrifuge without rubbing.* If Solution CD3 has precipitated, heat at 60°C until precipitate dissolves.

Perform all centrifugation steps at room temperature (15–25°C).

Steps

Prepare sample & Cell lysis

Spin the PowerBead Pro Tube briefly to ensure that the beads have settled at the bottom.

Add up to 250mg of soil and 800µL of Solution CD1. Vortex briefly to mix.

Note

After the sample has been loaded into the PowerBead Pro Tube, the next step is a homogenization and lysis procedure. The PowerBead Pro Tube contains a buffer that will (a) help disperse the soil particles, (b) begin to dissolve humic acids, and (c) protect nucleic acids from degradation. Gentle vortexing mixes the components in the PowerBead Pro Tube and begins to disperse the sample in the buffer.

Homogenize samples thoroughly using one of the following methods:

2.1.

Secure the PowerBead Pro Tube horizontally on a Vortex Adapter for 1.5–2 ml tubes (cat. no. 13000-V1-24). Vortex at maximum speed for 0h 10m 0s .

Note

If using Vortex Adapter for more than 12 preps simultaneously, increase the vortexing time by 5–10 min.

Note

Using the Vortex Adapter will maximize homogenization, which can lead to higher DNA yields. Avoid using tape, which can become loose and result in reduced homogenization efficiency, inconsistent results, and reduced yields.

2.2.

Use a PowerLyzer 24 Homogenizer. PowerBead Pro Tubes must be properly balanced in the tube holder of the PowerLyzer 24 Homogenizer. We recommend homogenizing the soil at 2000rpm , pausing for 0h 0m 30s , then homogenizing again at 2000rpm .

Note

Homogenizing samples at higher speeds (up to 4000 rpm) may increase yields but result in more fragmented DNA.

2.3.

Use a TissueLyser II. Place the PowerBead Pro Tube into the TissueLyser Adapter Set 2 x 24 (cat. no. 69982) or 2 ml Tube Holder (cat. no. 11993) and Plate Adapter Set (cat. no. 11990). Fasten the adapter into the instrument and shake for0h 5m 0s at speed 25 Hz. Reorient the adapter so that the side that was closest to the machine body is now furthest from it. Shake again for 5 min at a speed of 25 Hz.

Note

Vortexing/shaking is critical for complete homogenization and cell lysis. Cells are lysed by a combination of chemical agents from step 1 and mechanical shaking introduced at this step. Randomly shaking the beads in the presence of disruption agents will cause the beads to collide with microbial cells and lead to the cells breaking open.

Centrifuge the PowerBead Pro Tube at 15000rpm .

Transfer the supernatant to a clean 2 ml Microcentrifuge Tube (provided).

Note

Expect 500–600 µl. The supernatant may still contain some soil particles.

Inhibitor removal

Add 200µL of Solution CD2 and vortex for 0h 0m 5s .

Note

Solution CD2 contains IRT, which is a reagent that can precipitate non-DNA organic and inorganic material including humic substances, cell debris, and proteins. It is important to remove contaminating organic and inorganic matter that may reduce DNA purity and inhibit downstream DNA applications.

Centrifuge at 15000x g . Avoiding the pellet, transfer up to 700µL of supernatant to a clean 2 ml Microcentrifuge Tube (provided).

Note

Expect 500–600 µl.

Note

The pellet at this point contains non-DNA organic and inorganic material including humic acids, cell debris, and proteins. For best DNA yields and quality, avoid transferring any of the pellet.

Bind DNA

Add 600µL of Solution CD3 and vortex for 0h 0m 5s .

Note

Solution CD3 is a high-concentration salt solution. Because DNA binds tightly to silica at high salt concentrations, Solution CD3 will adjust the DNA solution salt concentration to allow binding of DNA, but not non-DNA organic and inorganic material that may still be present at low levels, to the MB Spin Column filter membrane.

Load 650µL of the lysate onto an MB Spin Column and centrifuge at 15000x g .

Note

DNA is selectively bound to the silica membrane in the MB Spin Column in the presence of high salt solution. Contaminants pass through the filter membrane, leaving only DNA bound to the membrane.

Discard the flow-through and repeat step 8 to ensure that all of the lysate has passed through the MB Spin Column.

10.

Carefully place the MB Spin Column into a clean 2 ml Collection Tube (provided). Avoid splashing any flow-through onto the MB Spin Column.

Wash

11.

.Add 500 µl of Solution EA to the MB Spin Column. Centrifuge at 15000x g .

Note

Solution EA is a wash buffer that removes protein and other non-aqueous contaminants from the MB Spin Column filter membrane.

12.

Discard the flow-through and place the MB Spin Column back into the same 2 ml Collection Tube.

13.

Add 500µL of Solution C5 to the MB Spin Column. Centrifuge at 15000x g .

Note

Solution C5 is an ethanol-based wash solution used to further clean the DNA that is bound to the silica filter membrane in the MB Spin Column. This wash solution removes residual salt, humic acid, and other contaminants while allowing the DNA to stay bound to the silica membrane.

14.

Discard the flow-through and place the MB Spin Column into a new 2 ml Collection Tube (provided).

15.

Centrifuge at up to 16000x g . Carefully place the MB Spin Column into a new 1.5 ml Elution Tube (provided).

Note

This spin removes residual Solution C5. It is critical to remove all traces of Solution C5 because the ethanol in it can interfere with downstream DNA applications, such as PCR, restriction digests, and gel electrophoresis.

Elute

16.

Add 50µL ~100µL of Solution C6 to the center of the white filter membrane.

Note

Placing Solution C6 in the center of the small white membrane will make sure the entire membrane is wet. This will result in a more efficient and complete release of the DNA from the MB Spin Column filter membrane. As Solution C6 passes through the silicamembrane, DNA that was bound in the presence of high salt is selectively released by Solution C6 (10 mM Tris), which lacks salt.

17.

Centrifuge at 15000x g . Discard the MB Spin Column. The DNA is now ready for downstream applications.

Note

We recommend storing the DNA frozen (–30 to –15°C or –90 to –65°C) as Solution C6 does not contain EDTA. To concentrate DNA, refer to the Troubleshooting Guide

QIAGEN® DNeasy® PowerSoil® Pro

Abstract

Before start

Steps

Prepare sample & Cell lysis

Inhibitor removal

Bind DNA

Wash

Elute

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