Qiagen DNeasy PowerSoil HTP 96 Kit
QIAGEN
Abstract
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 re
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.
Attachments
Steps
Sample preparation & cell lysis
REMOVE Spare Well Mat from a PowerBead Plate
ADD up to .25g of soil sample
ADD 750µL of PowerBead Solution to the wells of the PowerBead Plate
ADD 60µL of Solution C1
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 0h 10m 0s
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 0h 10m 0s
CENTRIFUGE at 4500x g for 0h 6m 0s at Room temperature
DISCARD the Square Well Mat
TRANSFER supernatant to a clean 1 mL Collection Plate
Inhibitor removal
ADD 250µL of Solution C2
APPLY Sealing Tape to the plate
VORTEX for 0h 0m 5s
INCUBATE at 2-8°C for 0h 10m 0s
CENTRIFUGE the plate at 4500x g for 0h 6m 0s at Room temperature
DISCARD Sealing Tape
AVOIDING the pellet, transfer the entire volume of supernatant to a new 1 mL Collection Plate
APPLY Sealing Tape to plate
VORTEX for 0h 0m 5s
INCUBATE at 2-8°C for 0h 10m 0s
CENTRIFUGE the plate at 4500x g for 0h 6m 0s at Room temperature
DISCARD Sealing Tape
AVOIDING the pellet, transfer the entire volume of supernatant to a new 1 mL Collection Plate
Bind DNA
ADD 200µL of Solution C3
REPEAT steps 7-8 once
APPLY Sealing Tape to the plate
CENTRIFUGE again at 4500x g for 0h 6m 0s at Room temperature
TRANSFER no more than 650µL of supernatant to a 2 mL Collection Plate
ADD 650µL of Solution C4 to each well of the plate
REPEAT to add 1300µL total
PIPET samples up and down to mix
PLACE a spin plate onto an S-Block
LOAD approximately 650µL into each well of the spin plate and seal the plate with an AirPore Tape Sheet
CENTRIFUGE at 4500x g for 0h 3m 0s atRoom temperature
DISCARD flow-through and place the spin plate back on the same S-Block
DISCARD AirPore Tape Sheet
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
Wash spin plate
ADD 500µL of Solution C5-D to each well of the spin plate and seal the plate with an AirPore Tape Sheet
CENTRIFUGE at 4500x g for 0h 3m 0s at Room temperature
DISCARD flow-through and place the spin plate back on the same S-Block
SEAL with an AirPore Tape Sheet
CENTRIFUGE again at 4500x g for 0h 5m 0s at Room temperature
DISCARD flow-through
CAREFULLY place the spin plate onto Racked Elution Microtubes
DISCARD AirPore Tape Sheet
ALLOW to air dry for 0h 10m 0s at Room temperature
Elute the DNA
ADD 100µL of Solution C6 to the center of each well
SEAL plate with an AirPore Tape Sheet
CENTRIFUGE at 4500x g,0h 0m 0s for 0h 3m 0s at Room temperature
DISCARD the AirPore Tape Sheet
SEAL Elution Microtubes with the Caps provided
DNA is now ready for downstream applications
