Dec 02, 2024
Version 3
Inhibitor-free DNA extraction from soil and sediment samples V.3
- 1University of Duisburg-Essen, Aquatic Ecosystem Research
- Aquatic Ecosystem Research
Protocol Citation: Dominik Buchner 2024. Inhibitor-free DNA extraction from soil and sediment samples. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6957zlqe/v3Version created by Dominik Buchner
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: December 02, 2024
Last Modified: December 02, 2024
Protocol Integer ID: 113427
Keywords: free dna extraction from soil, free dna from soil, dna extraction, free dna extraction, sediment samples this protocol, dneasy powermax soil kit, free dna, sediment sample, extraction, dna, sediment, less dna, soil, inhibitor, sample, miniaturized version
Abstract
This protocol describes how to extract inhibitor-free DNA from soil and sediment samples. 5 g of soil or up to 10 g of sediment can be processed in one extraction, but there is also a miniaturized version for 250 mg of input material, if less DNA is required. The protocol is based on the DNeasy PowerMax Soil Kit but costs much less. A lot of the buffers can be found in the following patent https://patents.google.com/patent/US7459548B2/en
Guidelines
Follow general lab etiquette. Wear gloves to prevent contaminating the samples. Clean the workspace before starting with 80% EtOH.
Materials
Materials required:
Below all materials needed for the protocol are listed. Vendors and part numbers are listed but interchangeable depending on the supply situation.
Chemicals:
Sodium phosphate dibasic Sodium phosphate dibasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #S0876-100G
Guanidinium thiocyanate Guanidinium thiocyanateFisher ScientificCatalog #10503345
Sodium phosphate monobasic Sodium phosphate monobasicSodium phosphate monobasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #S0751-100G
SDS ultrapure Sodium dodecyl sulfateDiagonalCatalog #A1112.0500
Sodium chloride Sodium chlorideFisher ScientificCatalog #10616082
Tris ultrapure 99.9% Tris ultrapure 99.9%DiagonalCatalog #A1086.1000
Hydrochloric acid fuming 37% Hydrochloric acid fuming 37%Merck MilliporeSigma (Sigma-Aldrich)Catalog #1003171011
Ammonium acetate Ammonium acetateCarl RothCatalog #7869.2
Aluminium ammonium sulfate dodecahydrateAluminium ammonium sulfate dodecahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A2140-500G
Guanidine hydrochloride Guanidine hydrochlorideFisher ScientificCatalog #10543325
Ethanol absolute Ethanol absoluteCarl RothCatalog #9065.4
Tween 20 Tween 20Carl RothCatalog #9127.1
Acetic acid Acetic acidCarl RothCatalog #7332.1
Ethanol absolute Ethanol absolute 99.8% p.a.Carl RothCatalog #9065.1
Labware:
50 mL centrifuge tubes, Ultra-High Performance Centrifuge tubes Ultra-High PerformanceVWR International (Avantor)Catalog #525-1098
Garnet Sharp Particles Garnet Sharp ParticlesBioSpec ProductsCatalog #11079103gar
Vortex Adapter for 2 (50 ml) tubesQiagenCatalog #13000-V1-50
Econospin Maxi Spin column EconoSpin® DNA Only Maxi Spin ColumnEpoch Life ScienceCatalog #2040-050
2 mL screwcap tubes 2 mL screwcap tubeSarstedtCatalog #72.693
The EconoSpin® All-In-One DNA Only Mini Spin Column The EconoSpin® All-In-One DNA Only Mini Spin ColumnEpoch Life ScienceCatalog #1920-250
Stock solutions:
1 L SDS stock solution 10 Mass / % volume
- Add 100 g SDS ultrapure to a beaker
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
1 L sodium chloride stock solution 5 Mass Percent
- Add 292.2 g sodium chloride to a beaker
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
1 L Tris stock solution 1 Mass Percent 8
- Add 121.14 g Tris ultrapure 99.9% to a beaker
- Adjust volume to 800 mL with ddH2O
- Adjust pH to 8 with HCl
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
500 mL sodium acetate stock solution 3 Molarity (M) 5
- Add 123 g sodium acetate to a beaker
- Adjust volume to 400 mL with ddH2O
- Adjust ph to 5 with acetic acid
- Adjust volume to 500 mL with ddH2O
- Sterilize by filtering and store at Room temperature
1 L Tris stock solution 1 Molarity (M) 7.5
- Add 121.14 g Tris ultrapure 99.9% to a beaker
- Adjust volume to 800 mL with ddH2O
- Adjust pH to 7.5 with HCl
- Adjust volume to 1 L with with ddH2O
- Sterilize by filtering and store at Room temperature
1 Molarity (M) 8.5
- Add 121.14 g Tris ultrapure 99.9% to a beaker
- Adjust volume to 800 mL with ddH2O
- Adjust pH to 8.5 with HCl
- Adjust volume to 1 L with with ddH2O
- Sterilize by filtering and store at Room temperature
1 L wash buffer stock solution (50 millimolar (mM) Tris ) 7.5
- Add 50 mL Tris stock solution 7.5 to a beaker
- Adjust volume to 1 L with with ddH2O
- Sterilize by filtering and store at Room temperature
Working solutions:
500 mL bead-beating solution (180 millimolar (mM) sodium phosphate , 120 millimolar (mM) guianidinium thiocyanate ) 8
- Add 12.8 g sodium phosphate dibasic to a beaker
- Add 7.1 g guanidinium thiocyanate
- Adjust volume to 490 mL with ddH2O
- Adjust pH to 8 by adding sodium phosphate monobasic
- Adjust volume to 500 mL with ddH2O
- Sterilize by filtering and store at Room temperature
500 mL lysis solution (150 millimolar (mM) sodium chloride , 4 Mass / % volume SDS , 500 millimolar (mM) Tris ) 8
- Add 200 mL of 10 Mass / % volume SDS stock solution to a beaker
- Add 15 mL of 5 Molarity (M) sodium chloride stock solution
- Add 250 mL of 1 Molarity (M) Tris stock solution 8
- Adjust volume to 500 mL with ddH2O
- Sterilize by filtering and store at Room temperature
500 mL ammonium acetate buffer (130 millimolar (mM) ammonium acetate )
- Add 5 g ammonium acetate to a beaker
- Adjust volume to 500 mL with ddH2O
- Sterilize by filtering and store at Room temperature
500 mL inhibitor removal solution (120 millimolar (mM) aluminum ammonium sulfate dodecahydrate )
- Add 27.2 g aluminium ammonium sulfate dodecahydrate to a beaker
- Adjust volume to 500 mL with ddH2O
- Sterilize by filtering and store at Room temperature
500 mL DNA binding buffer (2.5 Mass Percent Guanidine hydrochloride , 80 % (v/v) ethanol , 120 millimolar (mM) sodium acetate ) 5
- Add 119.4 g guanidine hydrochloride to a beaker
- Add 400 mL ethanol
- Add 20 mL 3 Molarity (M) sodium acetate stock solution 5
- Adjust volume to 500 mL with ddH2O
- Sterilize by filtering and store at Room temperature
1 L wash buffer (10 millimolar (mM) Tris , 80 % (v/v) Ethanol ) 7.5
- Add 200 mL was buffer stock solution
- Adjust volume to 1 L with Ethanol absolute
- Sterilize by filtering and store at Room temperature
1 L elution buffer (10 millimolar (mM) Tris ) 8.5
- Add 10 mL Tris stock solution 8.5 to a beaker
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
Safety warnings
Buffers containing guanidine produce highly reactive compounds when mixed with bleach. Don't mix the extraction waste with bleach or solutions that contain bleach.
Reagents are potentially damaging to the environment. Dispose waste as mandated.
Before start
Make sure all buffers are prepared before starting.
Protocol for up to 10 g of input material
20m 30s
Prepare one 50 mL centrifuge tube per sample with 15 g of garnet beads.
Add up to 10 g of soil to the tube.
Note
The amount of starting material differs from soil type to soil type. For most soil types 2 g of input material is sufficient. If the output is too low with 2 g it can be increased step by step.
Add 15 mL bead-beating solution and 1.2 mL lysis solution . Vortex shortly.
Place the samples on a Vortex adapter (e.g. Qiagen) and vortex at maximum speed for 00:10:00 .
Note
If you want to process more samples, instead of the vortex adapter a Thermoblock can be used. As an alternative, you can incubate the sample for 00:30:00 at 65 °C and at maximum RPM.
10m
2500 x g, 20°C, 00:03:00 . Transfer the supernatant to a new tube.
Note
For the large volume protocol, the samples can be carefully poured instead of being pipetted.
3m
Add 5 mL ammonium acetate buffer , vortex shortly, and incubate at 4 °C for 00:10:00 .
10m
2500 x g, 20°C, 00:04:00 . Transfer the supernatant to a new tube avoiding the pellet. The solution may still be colored, depending on the input material.
4m
Add 4 mL of inhibitor removal buffer . A precipitate may form. Vortex shortly, incubate at 4 °C for 00:10:00 .
10m
2500 x g, 20°C, 00:04:00 . The solution will clear up. Avoiding the pellet, transfer up to 15 mL to a new tube.
4m
Add 30 mL DNA binding buffer . Vortex or invert to mix.
Add the mixture to a maxi spin column (e.g. Epoch Life Science) in a 50 mL centrifuge tube.
2500 x g, 20°C, 00:00:30 . Discard the flow-through. Repeat once to bind the complete sample volume.
30s
Add 10 mL wash buffer . 2500 x g, 20°C, 00:05:00 to wash and dry the column.
5m
Transfer the column to a new tube. Add 1 mL elution buffer . Incubate for 00:03:00 at Room temperature .
3m
2500 x g, 20°C, 00:01:00 to elute the DNA. DNA eluate should be completely colorless and ready to go for downstream analysis.
1m
Protocol for up to 500 mg of input material
50m
Prepare one 2 mL centrifuge tube per sample with 750 mg of garnet beads.
Add 500 mg of soil or sediment sample.
Note
The optimal input is dependent of the input material used. For soils rich in organic material 200-250 mg input might be enough for sufficient output. For sediments mainly consisting of sand up to 1 g input may be used. Test the upper limit suitable for your sample type by increasing the input in small steps. If too much input is used it usually results in meager yields.
Add 1380 µL bead-beating solution and 240 µL lysis solution . Vortex shortly.
Place the samples on a Vortex adapter (e.g. Qiagen) and vortex at maximum speed for 00:10:00 .
Note
If you want to process more samples, instead of the vortex adapter a Thermoblock can be used. As an alternative, you can incubate the sample for 00:30:00 at 65 °C and at maximum RPM.
10m
10000 x g, 20°C, 00:03:00 . Transfer 600 µL of the supernatant to a new tube.
3m
Add 250 µL ammonium acetate buffer , vortex shortly, and incubate at 4 °C for 00:05:00 .
5m
10000 x g, 20°C, 00:01:00 . Transfer the supernatant to a new tube.
1m
Add 200 µL of inhibitor removal buffer . A precipitate may form. Vortex shortly, incubate at 4 °C for 00:05:00 .
5m
10000 x g, 20°C, 00:01:00 . Transfer 600 µL of the supernatant to a new tube.
1m
Add 1200 µL DNA binding buffer . Vortex to mix.
Load 650 µL of the mixture to a mini spin column (e.g. Epoch Life Science).
10000 x g, 20°C, 00:00:30 . Discard the flow-through. Repeat two times to bind the complete sample volume.
30s
Add 500 µL wash buffer . 10000 x g, 20°C, 00:00:30 to wash the column. Discard the flow-through.
30s
10000 x g, 20°C, 00:01:00 to dry the column. Transfer the spin column to a clean 1.5 mL microcentrifuge tube.
1m
Add 50 µL elution buffer . Incubate for 00:03:00 at Room temperature .
3m
10000 x g, 20°C, 00:01:00 to elute the DNA. DNA eluate should be completely colorless and ready to go for downstream analysis.
1m
Protocol references
The binding buffer from this protocol originates from the following protocol: