Sep 15, 2022
Low Biomass, high contamination Illumina DNA prep using DNeasy PowerSoil (Pro) Kit
- Tania Kurbessoian1,
- Jason E Stajich1,
- Sonia L. Ghose2
- 1UC Riverside;
- 2UC Davis
- Sonia L. Ghose: Originally adapted from.;
Protocol Citation: Tania Kurbessoian, Jason E Stajich, Sonia L. Ghose 2022. Low Biomass, high contamination Illumina DNA prep using DNeasy PowerSoil (Pro) Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr4ey3gmk/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 used this protocol on tar contaminated soil and worked great! Got good Illumina data for 16S and ITS1.
Created: September 15, 2022
Last Modified: September 15, 2022
Protocol Integer ID: 70110
Abstract
This is an addendum to the already optimized DNeasy PowerSoil Kit. This can also be applied to the PowerSoil Pro Kit.
Use this protocol for Low Biomass or high contamination samples.
Guidelines
Using some small tweaks to an already optimized protocol you can get your DNA from contaminated samples!
Materials
DNeasy Powersoil Kit (Adjusted name to PowerLyzer Kit) - https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/microbial-dna/dneasy-powerlyzer-powersoil-kit/
100% ethanol
Before start
Make sure you have all the components of the kit ready to go.
Add sample into the PowerBead tube.
Add 60μL of Solution C1.
Make sure the sample has not precipitated. If so, heat sample to 60ºC until precipitate is dissolved into solution.
Vortex to mix and incubate at 65ºC for 10 minutes.
10m
Bead beat at "homogenize" setting: 90 seconds bead beating, 60 seconds rest, then another 90 seconds (3 minutes total of actual beating).
4m
Centrifuge at 10,000 x g for 30 s.
30s
Remove the supernatant or upper aqueous layer if you have one, to the new tube.
Add 100 uL Solution C2 and 100μL of Solution C3. Vortex to mix. Incubate at 4ºC or on ice for 5 minutes.
5m
*This is for low humic soils (100μL of each). If high humic soil, add 150μL C2 followed by 150μL C3.
Centrifuge to pellet (1 min at 10,000 x g.) and transfer the supernatant to a new tube. Avoid pellet.
1m
Ideally you will have 650μL of lysate, and can add 650μL of Solution C4 and 650μL of 100% ethanol.
Make sure you mix C4 solution well. If new kit add ethanol.
If you have 700μL, add 700μL of Solution C4 and 600μL of 100% ethanol. (Keep it as close to 1:1 as possible)
Load the lysate to the spin filter 650μL at a time and bind in three steps, alternating with centrifugation (1 min at 10,000 x g). Discard flow-through each time.
3m
If the membrane is not stained brown, wash with 650μL of 100% ethanol (centrifuge 1 min at 10,000 x g and discard flow-through).
1m
If the membrane is stained brown, per sample, prepare a mix of 300μL of Solution C4 and 370μL of 100%
ethanol. Wash the column with this mixture first. Follow this wash with the 100% ethanol wash.
Wash with 500μL of solution C5 (centrifuge 1 min at 10,000 x g and discard flow-through)
1m
Dry the spin column for 2 minutes 10,000 x g. Transfer to a clean tube.
2m
Elute in 60μL of buffer C6 (Heat this buffer to 60ºC before eluting). Let the buffer sit on the membrane 5
minutes before elution.
5m
Then centrifuge for 1 min 10,000 x g into your storage tube.
1m