TITLE: QIAGEN DNeasy PowerSoil Pro Kit
AUTHORS: QIAGEN

[DESCRIPTION]
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 Unpublished studies report poor DNA yields and low concentrations of fish sedDNA.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. 

[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]
SECTION: Sample preparation &amp; cell lysis
1. SPIN the PowerBead Pro Tube briefly to ensure that the beads have settled at the bottom

ADD up to 0.25 g  of soil sample to the PowerBead Pro Tube

ADD 800 µL  of Solution CD1

VORTEX briefly to mix
SECTION: Sample preparation &amp; cell lysis
2. HOMOGENIZE samples thoroughly using one of the following methods:
SECTION: Inhibitor removal
4. ADD 200 µL  of Solution CD2

VORTEX for 5 s
SECTION: Sample preparation &amp; cell lysis
2.1. SECURE the PowerBead Pro Tube horizontally on a Vortex Adapter for 1.5–2 ml  tubes

VORTEX at maximum speed for 10 min
SECTION: Sample preparation &amp; cell lysis
2.2. USING a PowerLyzer 24 Homogenizer, homogenize the soil at 2000 rpm for  

PAUSE for 30 s

HOMOGENIZE again at 2000 rpm for 30 s 
SECTION: Sample preparation &amp; cell lysis
2.3. USING a TissueLyser II, place the PowerBead Pro Tube into the TissueLyser Adapter

FASTEN the adapter into the instrument and shake for 5 min  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.
SECTION: Sample preparation &amp; cell lysis
3. CENTRIFUGE the PowerBead Pro Tube at 15000 x g for 1 min 

TRANSFER supernatant to a clean 2 mL Microcentrifuge Tube (expect 500-600ul)
SECTION: Inhibitor removal
5. CENTRIFUGE tubes at 15000 x g for 1 min

AVOIDING the pellet, transfer up to 700 µL  of supernatant to a clean 2 ml Microcentrifuge Tube
SECTION: Bind DNA
6. ADD 600 µL  of Solution CD3

VORTEX for 5 s
SECTION: Bind DNA
7. LOAD 650 µL of the lysate onto an MB Spin Column

CENTRIFUGE at 15000 x g for 1 min 

DISCARD the liquid flow-through
SECTION: Bind DNA
8. 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
SECTION: Wash spin column
9. ADD 500 µL  of Solution EA to the MB Spin Column

CENTRIFUGE at 15000 x g for 1 min 

DISCARD the flow-through and place the MB Spin Column into the same 2 mL Collection Tube
SECTION: Wash spin column
10. ADD 500 µL  of Solution C5 to the MB Spin Column

CENTRIFUGE at 15000 x g for 1 min 

DISCARD the flow-through and place the MB Spin Column into a NEW 2 mL Collection Tube
SECTION: Wash spin column
11. CENTRIFUGE at 16000 x g for 2 min

CAREFULLY place the MB Spin Column into a new 1.5 ml Elution Tube
SECTION: Elute the DNA
12. ADD between 50 µL and 100 µL of Solution C6 to the center of the white filter membrane

CENTRIFUGE at 15000 x g for 1 min 

DISCARD the MB Spin Column

DNA is now ready for downstream applications