Oct 30, 2023
Quick-Start Protocol for DNeasy® PowerSoil® Pro Kit
- 1NC State University
Protocol Citation: Carlos Carlos Goller 2023. Quick-Start Protocol for DNeasy® PowerSoil® Pro Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkwk45l5r/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: Working
We use this protocol and it's working
Created: October 18, 2022
Last Modified: February 28, 2024
Protocol Integer ID: 71497
Keywords: PowerSoil, metagenomics, soil
Abstract
This protocol will allow users to operate the DNeasy® PowerSoil® Pro Kit.
Guidelines
Solution CD2 should be stored at 2–8°C upon arrival. All other reagents and kit components should be stored at room temperature (15–25°C).
Further information
- DNeasy® PowerSoil® Pro Kit Handbook: www.qiagen.com/HB-2495
- Safety Data Sheets: www.qiagen.com/safety
- Technical assistance: support.qiagen.com
Materials
The DNeasy® PowerSoil® Pro Kit. All requirements included within.
Safety warnings
Use caution when operating the vortex. Ensure tubes are balanced before turning on the instrument. Dispose of materials in appropriate waste containers.
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 the precipitate dissolves.
- Perform all centrifugation steps at room temperature (15–25°C).
Using the DNeasy® PowerSoil® Pro Kit
Using the DNeasy® PowerSoil® Pro Kit
18m 10s
Spin the PowerBead Pro Tube briefly to ensure that the beads have settled at the bottom. Add up to 250 mg of soil and 800 µL of Solution CD1. Vortex briefly to mix.
Secure the PowerBead Pro Tube horizontally on a Vortex Adapter for 1.5–2 ml tubes (cat. no. 13000-V1-24). Vortex at maximum speed for 00:10:00
Note
If using the Vortex Adapter for more than 12 preps simultaneously, increase the vortexing time by 5-10 min
For more information about other bead beating methods, see the “Protocol: Detailed” section of DNeasy® PowerSoil® Pro Kit Handbook.
10m
Centrifuge the PowerBead Pro Tube at 15000 x g for 00:01:00
1m
Transfer the supernatant to a clean 2 ml Microcentrifuge Tube (provided)
Note
Expect 500–600 µl. The supernatant may still contain some soil particles
Add 200 µL of Solution CD2 and vortex for 00:00:05
5s
Centrifuge at 15000 x g for 00:01:00 at Room temperature . Avoiding the pellet, transfer up to 700 µL of supernatant to a clean 2 ml Microcentrifuge Tube (provided)
Note
Expect 500–600 µl.
1m
Add 600 µL of Solution CD3 and vortex for 00:00:05 .
5s
Load 650 µL of the lysate onto an MB Spin Column and centrifuge at 15000 x g for 00:01:00
1m
Discard the flow-through and repeat step 8 to ensure that all of the lysate has passed through the MB Spin Column.
0. Carefully place the MB Spin Column into a clean 2 ml Collection Tube (provided).
Safety information
Avoid splashing any flow-through onto the MB Spin Column
Add 500 µL of Solution EA to the MB Spin Column. Centrifuge at 15000 x g for 00:01:00
1m
. Discard the flow-through and place the MB Spin Column back into the same 2 ml Collection Tube.
Add 500 µL of Solution C5 to the MB Spin Column. Centrifuge at 15000 x g for 00:01:00
1m
Discard the flow-through and place the MB Spin Column into a new 2 ml Collection Tube (provided).
Centrifuge at up to 16000 x g for 00:02:00 . Carefully place the MB Spin Column into a new 1.5 ml Elution Tube (provided)
2m
. Add 50 mL -100 mL of Solution C6 to the center of the white filter membrane.
Centrifuge at 15000 x g for 00:01:00 . Discard the MB Spin Column. The DNA is now ready for downstream applications
Note
We recommend storing the DNA frozen (–30 to –15°C or –90 to –65°C) as Solution C6 does not contain EDTA. To concentrate DNA, please refer to the Troubleshooting Guide.
1m