Sep 10, 2022

Public workspaceQIAGEN® DNeasy® PowerSoil® Pro V.1

DOI
dx.doi.org/10.17504/protocols.io.bp2l69411lqe/v1
  • 1u109023016@gap.kmu.edu.tw
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DOI: dx.doi.org/10.17504/protocols.io.bp2l69411lqe/v1
External link: https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/microbial-dna/dneasy-powersoil-pro-kit/
Protocol Citation: Hsin-Mao Wu 2022. QIAGEN® DNeasy® PowerSoil® Pro. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l69411lqe/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: In development
We are still developing and optimizing this protocol
Created: September 10, 2022
Last Modified: September 13, 2023
Protocol Integer ID: 69796
Abstract
For the isolation of microbial genomic DNA from all soil types, including difficult samples such as compost, sediment, and manure.
Guidelines


Materials
  1. PowerBead Pro Tubes
  2. MB Spin Columns
  3. Solution CD1
  4. Solution CD2
  5. Solution CD3
  6. Solution EA
  7. Solution C5
  8. Solution C6
  9. Microcentrifuge Tubes (2 ml)
  10. Elution Tubes (1.5 ml)
  11. Collection Tubes (2 ml)
  12. Microcentrifuge
  13. Pipettor
Safety warnings
  1. Solution EA and Solution C5 are flammable
  2. DO NOT add bleach or acidic solutions directly to the sample preparation waste.
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).
Prepare sample & Cell lysis
Prepare sample & Cell lysis
Spin the PowerBead Pro Tube briefly to ensure that the beads have settled at the bottom.
Add up to Amount250 mg of soil and Amount800 µL 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:
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 Duration00:10:00 .
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.


10m
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 Centrifigation2000 rpm, 00:00:30 , pausing for Duration00:00:30 , then homogenizing again at Centrifigation2000 rpm, 00:00:30 .

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

1m 30s
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 forDuration00:05:00 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.

5m
Centrifuge the PowerBead Pro Tube at Centrifigation15000 rpm, 00:01:00 .

1m
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
Inhibitor removal
1m 5s
1m 5s
Add Amount200 µL of Solution CD2 and vortex for Duration00:00:05 .
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.


5s
Centrifuge at Centrifigation15000 x g, 00:01:00 . Avoiding the pellet, transfer up to Amount700 µL 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.

1m
Bind DNA
Bind DNA
1m 5s
1m 5s
Add Amount600 µL of Solution CD3 and vortex for Duration00:00:05 .
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.


5s
Load Amount650 µL of the lysate onto an MB Spin Column and centrifuge at Centrifigation15000 x g, 00:01:00 .
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.


1m
Discard the flow-through and repeat step 8 to ensure that all of the lysate has passed through the MB Spin Column.
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
Wash
4m
4m
.Add 500 µl of Solution EA to the MB Spin Column. Centrifuge at Centrifigation15000 x g, 00:01:00 .
Note
Solution EA is a wash buffer that removes protein and other non-aqueous contaminants from the MB Spin Column filter membrane.


1m
Discard the flow-through and place the MB Spin Column back into the same 2 ml Collection Tube.
Add Amount500 µL of Solution C5 to the MB Spin Column. Centrifuge at Centrifigation15000 x g, 00:01:00 .
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.


1m
Discard the flow-through and place the MB Spin Column into a new 2 ml Collection Tube (provided).
Centrifuge at up to Centrifigation16000 x g, 00:02:00 . 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.


2m
Elute
Elute
1m
1m
Add Amount50 µL ~Amount100 µL 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 silica
membrane, DNA that was bound in the presence of high salt is selectively released by
Solution C6 (10 mM Tris), which lacks salt.


Centrifuge at Centrifigation15000 x g, 00:01:00 . 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


1m