Apr 07, 2023

Public workspaceQIAGEN DNeasy PowerSoil Pro Kit

 Forked from QIAGEN® DNeasy® PowerSoil® Pro
DOI
dx.doi.org/10.17504/protocols.io.kxygx97mzg8j/v1
  • QIAGEN1
  • 1QIAGEN
Yuanyu Cheng
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DOI: dx.doi.org/10.17504/protocols.io.kxygx97mzg8j/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: QIAGEN 2023. QIAGEN DNeasy PowerSoil Pro Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx97mzg8j/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: Other
No published studies (as of April 2023) using this protocol to detect fish sedDNA Unpublished studies on lake surface sediments report low detection of fish sedDNA Protocol successful at detecting fish sedDNA collected in a stream during a fish migration
Created: December 21, 2022
Last Modified: April 18, 2023
Protocol Integer ID: 74332
Keywords: Qiagen, DNeasy, PowerSoil Pro, SedDNA, Sedimentary DNA, Fish
Abstract
For the isolation of microbial genomic DNA from all soil types, including difficult samples such as compost, sediment, and manure.

The DNeasy PowerSoil Pro Kit comprises a novel and proprietary method for isolating microbial genomic DNA from environmental samples. The kit uses QIAGEN’s secondgeneration Inhibitor Removal Technology® (IRT) and 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 and stool types have also been used successfully with this kit. Improved IRT combined with more efficient bead beating and lysis chemistry yields high-quality DNA that can be used immediately in downstream applications, including PCR, qPCR, and next-generation sequencing (16S and whole genome).

As of April, 2023 - no published studies successfully detecting fish sedDNA using only this kit (see Lakes ABPS protocol). Unpublished studies report poor DNA yields and low concentrations of fish sedDNA. Other studies targeting migratory fish sedDNA during a spawning run found sufficient fish sedDNA concentrations following this protocol.
Guidelines


Materials
DNeasy PowerSoil Pro Kit (50 preparations) already includes:
  • PowerBead Pro Tubes * 50
  • MB Spin Columns * 50
  • Solution CD1 Amount40 mL
  • Solution CD2 Amount15 mL
  • Solution CD3 Amount35 mL
  • Solution EA Amount36 mL
  • Solution C5 Amount30 mL
  • Solution C6 Amount9 mL
  • Microcentrifuge Tubes (2 mL) * 100
  • Elution Tubes (1.5 mL) * 50
  • Collection Tubes (2 mL) * 100

Equipment and Reagents to be Supplied by User
  • Microcentrifuge (up to 16,000 x g)
  • Pipettor (50-1000 ul)
  • Vortex Genie
  • Vortex Adapter for 24 (1.5-2 mL) tubes






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).
Sample preparation & cell lysis
Sample preparation & cell lysis
SPIN the PowerBead Pro Tube briefly to ensure that the beads have settled at the bottom

ADD up to Amount0.25 g of soil sample to the PowerBead Pro Tube

ADD Amount800 µL of Solution CD1

VORTEX briefly to mix
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

VORTEX at maximum speed for Duration00:10:00
10m
USING a PowerLyzer 24 Homogenizer, homogenize the soil at Centrifigation2000 rpm for Duration00:00:00

PAUSE for Duration00:00:30

HOMOGENIZE again at Centrifigation2000 rpm for Duration00:00:30
1m
USING a TissueLyser II, place the PowerBead Pro Tube into the TissueLyser Adapter

FASTEN the adapter into the instrument and shake for Duration00:05:00 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 Duration00:05:00 at a speed of 25 Hz.
10m
CENTRIFUGE the PowerBead Pro Tube at Centrifigation15000 x g for Duration00:01:00

TRANSFER supernatant to a clean 2 mL Microcentrifuge Tube (expect 500-600ul)
1m
Inhibitor removal
Inhibitor removal
1m 5s
1m 5s
ADD Amount200 µL of Solution CD2

VORTEX for Duration00:00:05
5s
CENTRIFUGE tubes at Centrifigation15000 x g for Duration00:01:00

AVOIDING the pellet, transfer up to Amount700 µL of supernatant to a clean 2 ml Microcentrifuge Tube
1m
Bind DNA
Bind DNA
1m 5s
1m 5s
ADD Amount600 µL of Solution CD3

VORTEX for Duration00:00:05
5s
LOAD Amount650 µL of the lysate onto an MB Spin Column

CENTRIFUGE at Centrifigation15000 x g for Duration00:01:00

DISCARD the liquid flow-through
1m
REPEAT step 7 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. Avoid splashing any flow-through onto the MB Spin Column
Wash spin column
Wash spin column
4m
4m
ADD Amount500 µL of Solution EA to the MB Spin Column

CENTRIFUGE at Centrifigation15000 x g for Duration00:01:00

DISCARD the flow-through and place the MB Spin Column into the same 2 mL Collection Tube
1m
ADD Amount500 µL of Solution C5 to the MB Spin Column

CENTRIFUGE at Centrifigation15000 x g for Duration00:01:00

DISCARD the flow-through and place the MB Spin Column into a NEW 2 mL Collection Tube
1m
CENTRIFUGE at Centrifigation16000 x g for Duration00:02:00

CAREFULLY place the MB Spin Column into a new 1.5 ml Elution Tube
2m
Elute the DNA
Elute the DNA
1m
1m
ADD between Amount50 µL and Amount100 µL of Solution C6 to the center of the white filter membrane

CENTRIFUGE at Amount15000 x g for Duration00:01:00

DISCARD the MB Spin Column

DNA is now ready for downstream applications
1m
Protocol references