TITLE: Qiagen DNeasy PowerSoil HTP 96 Kit
AUTHORS: QIAGEN

[DESCRIPTION]
Introduction 
The DNeasy PowerSoil HTP 96 Kit allows high-throughput isolation of DNA from up to 384 soil samples in less than one day. 

Principle and procedure 
This kit provides researchers with a high-throughput method for isolating genomic DNA from environmental samples using Inhibitor Removal Technology® (IRT) that efficiently removes humic substances that inhibit PCR. This procedure effectively removes PCR inhibitors from even the most difficult soil types, allowing for more successful PCR amplification of DNA. DNA isolated from many sample types, including compost, sediment and manure, was successfully used as template to amplify members of a wide range of microbial groups in soils. These include bacteria (gram-positive, gram-negative and spore-formers), actinomycetes, archaebacteria and fungi. 
Environmental samples are added to a 96 well bead beating plate for rapid and thorough homogenization. Cell lysis occurs by a combination of mechanical and chemical methods. Humic substances are removed by a specialized precipitation process. Total genomic DNA is captured on a 96 well silica membrane in a spin-column plate format. DNA is then washed and eluted from the membrane. The eluted DNA is ready for PCR analysis and other downstream applications. 
The estimated time from start to finish to process two 96 well plates for this protocol is approximately 8 hours. Stopping points at appropriate steps are mentioned in the protocol. The majority of the time is for weighing and loading the soil samples into the 96 well plates. 

As of April, 2023 - no published studies successfully detecting fish sedDNA using this protocol, however research is still in development and we hope to see promising results in the future

[BEFORE_START]
Important points before starting:
If Solution C1 has precipitated, heat at 60°C until precipitate dissolves. 
Prepare solution C5-D by adding an equal volume (120 ml) of 100% ethanol. Mix well. 

[GUIDELINES]
The DNeasy PowerSoil HTP 96 Kit reagents and components can be stored at room temperature (15–25°C) until the expiry date printed on the box label. 

This DNeasy PowerSoil HTP 96 Kit is designed to process 0.25 g of soil.  Recommended starting amounts for different soil types are listed in Table 1.

[STEPS]
SECTION: Sample preparation &amp; cell lysis
2. ADD 750 µL of PowerBead Solution to the wells of the PowerBead Plate

ADD 60 µL of Solution C1
SECTION: Sample preparation &amp; cell lysis
3. SECURE the Square Well Mat tightly to the plate

PLACE PowerBead Plate with the mat securely fastened between 2 Adapter Plates on a 96 well plate shaker or a TissueLyzer II

SHAKE at speed 20 Hz for 10 min 

RE-ORIENT plates so the side that was closest to the machine body is now furthest from it and shake again at speed 20 Hz for 10 min 
SECTION: Sample preparation &amp; cell lysis
4. CENTRIFUGE at 4500 x g for 6 min at Room temperature 

DISCARD the Square Well Mat

TRANSFER supernatant to a clean 1 mL Collection Plate
 

SECTION: Inhibitor removal
5. ADD 250 µL of Solution C2

APPLY Sealing Tape to the plate

VORTEX for 5 s 

INCUBATE at 2-8 °C for 10 min 
 

SECTION: Inhibitor removal
6. CENTRIFUGE the plate at 4500 x g for 6 min at Room temperature 

DISCARD Sealing Tape

AVOIDING the pellet, transfer the entire volume of supernatant to a new 1 mL Collection Plate
SECTION: Inhibitor removal
7. APPLY Sealing Tape to plate

VORTEX for 5 s 

INCUBATE at 2-8 °C for 10 min 
SECTION: Inhibitor removal
8. CENTRIFUGE the plate at 4500 x g for 6 min at Room temperature 

DISCARD Sealing Tape

AVOIDING the pellet, transfer the entire volume of supernatant to a new 1 mL Collection Plate
SECTION: Bind DNA
9. ADD 200 µL of Solution C3

REPEAT steps 7-8 once
SECTION: Bind DNA
11. ADD 650 µL of Solution C4 to each well of the plate

REPEAT to add 1300 µL total
 


SECTION: Bind DNA
12. PIPET samples up and down to mix

PLACE a spin plate onto an S-Block
SECTION: Bind DNA
13. LOAD approximately 650 µL into each well of the spin plate and seal the plate with an AirPore Tape Sheet

CENTRIFUGE at 4500 x g for 3 min atRoom temperature 

DISCARD flow-through and place the spin plate back on the same S-Block

DISCARD AirPore Tape Sheet
SECTION: Bind DNA
14. REPEAT step 13 until all the supernatant has been processed

DISCARD the final flow-through

PLACE the spin plate back on the same S-Block
SECTION: Wash spin plate
15. ADD 500 µL of Solution C5-D to each well of the spin plate and seal the plate with an AirPore Tape Sheet

CENTRIFUGE at 4500 x g for 3 min at Room temperature 

DISCARD flow-through and place the spin plate back on the same S-Block

SEAL with an AirPore Tape Sheet
SECTION: Wash spin plate
16. CENTRIFUGE again at 4500 x g for 5 min at Room temperature 

DISCARD flow-through

CAREFULLY place the spin plate onto Racked Elution Microtubes

DISCARD AirPore Tape Sheet
SECTION: Wash spin plate
17. ALLOW to air dry for 10 min at Room temperature
SECTION: Elute the DNA
18. ADD 100 µL of Solution C6 to the center of each well 

SEAL plate with an AirPore Tape Sheet
SECTION: Elute the DNA
19. CENTRIFUGE at 4500 x g for 3 min at Room temperature 

DISCARD the AirPore Tape Sheet
SECTION: Elute the DNA
20. SEAL Elution Microtubes with the Caps provided

DNA is now ready for downstream applications
 


SECTION: Sample preparation &amp; cell lysis
1. REMOVE Spare Well Mat from a PowerBead Plate

ADD up to .25 g of soil sample
 

SECTION: Bind DNA
10. APPLY Sealing Tape to the plate

CENTRIFUGE again at 4500 x g for 6 min at Room temperature 

TRANSFER no more than 650 µL of supernatant to a 2 mL Collection Plate