Apr 13, 2023
Qiagen DNeasy PowerSoil HTP 96 Kit
- QIAGEN 1
- 1QIAGEN
Protocol Citation: QIAGEN 2023. Qiagen DNeasy PowerSoil HTP 96 Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5j526l1b/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: March 20, 2023
Last Modified: April 18, 2023
Protocol Integer ID: 79105
Keywords: isolating genomic dna, genomic dna from environmental sample, microbial groups in soil, successful pcr amplification of dna, throughput isolation of dna, soil sample, humic substance, genomic dna, eluted dna, soil, pcr amplification, difficult soil type, dna, total genomic dna, dneasy powersoil htp 96 kit introduction, successful pcr amplification, microbial group, dneasy powersoil htp 96 kit, pcr analysis, environmental sample, ready for pcr analysis, bacteria, pcr inhibitor, fungi, qiagen dneasy powersoil htp 96 kit introduction, qiagen dneasy powersoil htp
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
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
Attachments
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.
Materials
Equipment and Reagents to Be Supplied by User
- Centrifuge capable of handling two 96 well blocks (13 cm x 8.5 cm x 60 cm) at 4500 x g Note: If you have a centrifuge with a maximum speed less than 4500 x g, see the Troubleshooting Guide.
- Multi-channel pipettor (50–650 μl)
- Mechanical shaker for 96 well blocks and plate adaptors (cat. no. 11990)
- Vortex-Genie® 2 vortex with 3-inch platform
- 100% ethanol
- Reagent reservoirs (optional)
- Vacuum pump (optional)
- Vacuum manifold (optional)
- Plate seals (optional – if protocol is paused at step 15)
Kit Contents (384 preps; 12955-4)
- QIAamp® 96 Plates (4)
- PowerBead DNA Plates, Garnet (4)
- PowerBead Solution (2 x 200 ml)
- Solution C1 (45 ml)
- Solution C2 (128 ml)
- Solution C3 (106 ml)
- Solution C4 (2 x 330 ml)
- Solution C5-D* (120 ml)
Note: *Before using for the first time, add 100% ethanol to Solution C5-D, as indicated in the protocol, to obtain a working solution.
- Solution C6 (66 ml)
- Racked Elution Microtubes (4)
- Caps for Elution Microtubes (50 x 8)
- Collection Plates, 1 ml (4 x 4)
- Collection Plates, 2 ml (4)
- AirPore Tape Sheets (25)
- Sealing Tape, Polyester, 2 ml (16)
- S-Blocks (4)
- Square Well Mats (4)
- Quick Start Protocol (1)
Troubleshooting
Safety warnings
When working with chemicals, always wear a suitable lab coat, disposable gloves and protective goggles. For more information, please consult the appropriate safety data sheets (SDSs). These are available online in convenient and compact PDF format at www.qiagen.com/safety where you can find, view and print the SDS for each QIAGEN kit and kit component.
Solution C5-D is flammable after addition of ethanol.
DO NOT add bleach or acidic solutions directly to the sample preparation waste.
PowerBead Solution and Solution C4 contain guanidine salts, which can form highly reactive compounds when combined with bleach. If liquid containing these buffers is spilt, clean with a suitable laboratory detergent and water. If the spilt liquid contains potentially infectious agents, clean the affected area first with laboratory detergent and water, and then with 1% (v/v) sodium hypochlorite.
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.
Sample preparation & cell lysis
1h 12m 5s
REMOVE Spare Well Mat from a PowerBead Plate
ADD up to .25 g of soil sample
Note
Avoid cross contamination between sample wells. This is an appropriate stopping point. PowerBead Plate can be stored at 2-8 °C covered with the Square Well Mat
ADD 750 µL of PowerBead Solution to the wells of the PowerBead Plate
ADD 60 µL 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 00:10:00
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 00:10:00
20m
CENTRIFUGE at 4500 x g for 00:06:00 at Room temperature
DISCARD the Square Well Mat
TRANSFER supernatant to a clean 1 mL Collection Plate
Note
Supernatant may still contain some soil particles
6m
Inhibitor removal
1h 12m 5s
ADD 250 µL of Solution C2
APPLY Sealing Tape to the plate
VORTEX for 00:00:05
INCUBATE at 2-8 °C for 00:10:00
Note
You can skip the 10 min incubation. However, if you have already validated DNeasy PowerSoil extractions with the incubation, it is recommended to retain this step
10m 5s
CENTRIFUGE the plate at 4500 x g for 00:06:00 at Room temperature
DISCARD Sealing Tape
AVOIDING the pellet, transfer the entire volume of supernatant to a new 1 mL Collection Plate
6m
APPLY Sealing Tape to plate
VORTEX for 00:00:05
INCUBATE at 2-8 °C for 00:10:00
10m 5s
CENTRIFUGE the plate at 4500 x g for 00:06:00 at Room temperature
DISCARD Sealing Tape
AVOIDING the pellet, transfer the entire volume of supernatant to a new 1 mL Collection Plate
6m
Bind DNA
30m
ADD 200 µL of Solution C3
REPEAT steps 7-8 once
APPLY Sealing Tape to the plate
CENTRIFUGE again at 4500 x g for 00:06:00 at Room temperature
TRANSFER no more than 650 µL of supernatant to a 2 mL Collection Plate
6m
ADD 650 µL of Solution C4 to each well of the plate
REPEAT to add 1300 µL total
Note
You can pause here and store samples covered with Sealing Tape at 2-8 °C
PIPET samples up and down to mix
PLACE a spin plate onto an S-Block
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 00:03:00 atRoom temperature
DISCARD flow-through and place the spin plate back on the same S-Block
DISCARD AirPore Tape Sheet
3m
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
30m
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 00:03:00 at Room temperature
DISCARD flow-through and place the spin plate back on the same S-Block
SEAL with an AirPore Tape Sheet
3m
CENTRIFUGE again at 4500 x g for 00:05:00 at Room temperature
DISCARD flow-through
CAREFULLY place the spin plate onto Racked Elution Microtubes
DISCARD AirPore Tape Sheet
5m
ALLOW to air dry for 00:10:00 at Room temperature
10m
Elute the DNA
30m
ADD 100 µL of Solution C6 to the center of each well
SEAL plate with an AirPore Tape Sheet
CENTRIFUGE at 4500 x g for 00:03:00 at Room temperature
DISCARD the AirPore Tape Sheet
3m
SEAL Elution Microtubes with the Caps provided
DNA is now ready for downstream applications
Note
Qiagen recommends storing DNA frozen (-15 °C to 30 °C or -65 °C to 90 °C ) as Solution C6 does not contain EDTA