Nov 29, 2023

Public workspaceInhibitor-free DNA extraction from soil and sediment samples V.2

DOI
dx.doi.org/10.17504/protocols.io.bp2l6957zlqe/v2
  • 1University of Duisburg-Essen, Aquatic Ecosystem Research
Dominik Buchner
Open access
DOI: dx.doi.org/10.17504/protocols.io.bp2l6957zlqe/v2
Protocol Citation: Dominik Buchner 2023. Inhibitor-free DNA extraction from soil and sediment samples. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6957zlqe/v2Version 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: November 29, 2023
Last Modified: November 29, 2023
Protocol Integer ID: 91569
Abstract
This protocol describes how to extract inhibitor-free DNA from soil and sediment samples. Amount5 g of soil or up to Amount10 g of sediment can be processed in one extraction, but there is also a miniaturized version for Amount250 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 ReagentSodium phosphate dibasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #S0876-100G
Guanidinium thiocyanate ReagentGuanidinium thiocyanateFisher ScientificCatalog #10503345
Sodium phosphate monobasic Sodium phosphate monobasicReagentSodium phosphate monobasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #S0751-100G
SDS ultrapure ReagentSodium dodecyl sulfateDiagonalCatalog #A1112.0500
Sodium chloride ReagentSodium chlorideFisher ScientificCatalog #10616082
Tris ultrapure 99.9% ReagentTris ultrapure 99.9%DiagonalCatalog #A1086.1000
Hydrochloric acid fuming 37% ReagentHydrochloric acid fuming 37%Merck MilliporeSigma (Sigma-Aldrich)Catalog #1003171011
Ammonium acetate ReagentAmmonium acetateCarl RothCatalog #7869.2
Aluminium ammonium sulfate dodecahydrateReagentAluminium ammonium sulfate dodecahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A2140-500G
Guanidine hydrochloride ReagentGuanidine hydrochlorideFisher ScientificCatalog #10543325
Isopropanol Reagent2-PropanolCarl RothCatalog #1HPK.1
Tween 20 ReagentTween 20Carl RothCatalog #9127.1
Acetic acid ReagentAcetic acidCarl RothCatalog #7332.1
Ethanol absolute ReagentEthanol absolute 99.8% p.a.Carl RothCatalog #9065.1



Labware:
50 mL centrifuge tubes, Ultra-High Performance ReagentCentrifuge tubes Ultra-High PerformanceVWR InternationalCatalog #525-1098
Garnet Sharp Particles ReagentGarnet Sharp ParticlesBioSpec ProductsCatalog #11079103gar
ReagentVortex Adapter for 2 (50 ml) tubesQiagenCatalog #13000-V1-50
Econospin Maxi Spin column ReagentEconoSpin® DNA Only Maxi Spin ColumnEpoch Life ScienceCatalog #2040-050
2 mL screwcap tubes Reagent2 mL screwcap tubeSarstedtCatalog #72.693
The EconoSpin® All-In-One DNA Only Mini Spin Column ReagentThe EconoSpin® All-In-One DNA Only Mini Spin ColumnEpoch Life ScienceCatalog #1920-250


Stock solutions:
Amount1 L SDS stock solution Concentration10 Mass / % volume
  • Add Amount100 g SDS ultrapure to a beaker
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L sodium chloride stock solution Concentration5 Molarity (M)
  • Add Amount292.2 g sodium chloride to a beaker
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L Tris stock solution Concentration1 Molarity (M) Ph8
  • Add Amount121.14 g Tris ultrapure 99.9% to a beaker
  • Adjust volume to Amount800 mL with ddH2O
  • Adjust pH to Ph8 with HCl
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount500 mL sodium acetate stock solution Concentration3 Molarity (M) Ph5
  • Add Amount123 g sodium acetate to a beaker
  • Adjust volume to Amount400 mL with ddH2O
  • Adjust ph to Ph5 with acetic acid
  • Adjust volume to Amount500 mL with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L Tris stock solution Concentration1 Molarity (M) Ph7.5
  • Add Amount121.14 g Tris ultrapure 99.9% to a beaker
  • Adjust volume to Amount800 mL with ddH2O
  • Adjust pH to Ph7.5 with HCl
  • Adjust volume to Amount1 L with with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Concentration1 Molarity (M) Ph8.5
  • Add Amount121.14 g Tris ultrapure 99.9% to a beaker
  • Adjust volume to Amount800 mL with ddH2O
  • Adjust pH to Ph8.5 with HCl
  • Adjust volume to Amount1 L with with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L wash buffer stock solution (Concentration50 millimolar (mM) Tris ) Ph7.5
  • Add Amount50 mL Tris stock solution Ph7.5 to a beaker
  • Adjust volume to Amount1 L with with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Working solutions:
Amount500 mL bead-beating solution (Concentration180 millimolar (mM) sodium phosphate , Concentration120 millimolar (mM) guianidinium thiocyanate ) Ph8
  • Add Amount12.8 g sodium phosphate dibasic to a beaker
  • Add Amount7.1 g guanidinium thiocyanate
  • Adjust volume to Amount490 mL with ddH2O
  • Adjust pH to Ph8 by adding sodium phosphate monobasic
  • Adjust volume to Amount500 mL with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount500 mL lysis solution (Concentration150 millimolar (mM) sodium chloride , Concentration4 Mass / % volume SDS , Concentration500 millimolar (mM) Tris ) Ph8
  • Add Amount200 mL of Concentration10 Mass / % volume SDS stock solution to a beaker
  • Add Amount15 mL of Concentration5 Molarity (M) sodium chloride stock solution
  • Add Amount250 mL of Concentration1 Molarity (M) Tris stock solution Ph8
  • Adjust volume to Amount500 mL with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount500 mL ammonium acetate buffer (Concentration130 millimolar (mM) ammonium acetate )
  • Add Amount5 g ammonium acetate to a beaker
  • Adjust volume to Amount500 mL with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount500 mL inhibitor removal solution (Concentration120 millimolar (mM) aluminum ammonium sulfate dodecahydrate )
  • Add Amount27.2 g aluminium ammonium sulfate dodecahydrate to a beaker
  • Adjust volume to Amount500 mL with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount500 mL DNA binding buffer (Concentration5 Molarity (M) Guanidine hydrochloride , Concentration40 % (v/v) isopropanol , Concentration0.05 % (v/v) Tween 20 , Concentration115 millimolar (mM) sodium acetate ) Ph5
  • Add Amount238.8 g guanidine hydrochloride to a beaker
  • Add Amount200 mL isopropanol
  • Add Amount250 µL Tween 20
  • Add Amount20 mL Concentration3 Molarity (M) sodium acetate stock solution Ph5
  • Adjust volume to Amount500 mL with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L wash buffer (Concentration10 millimolar (mM) Tris , Concentration80 % (v/v) Ethanol ) Ph7.5
  • Add Amount200 mL was buffer stock solution
  • Adjust volume to Amount1 L with Ethanol absolute
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L elution buffer (Concentration10 millimolar (mM) Tris ) Ph8.5
  • Add Amount10 mL Tris stock solution Ph8.5 to a beaker
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature














Safety warnings
Attention
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
Protocol for up to 10 g of input material
20m 30s
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 Amount15 mL bead-beating solution and Amount1.2 mL lysis solution . Vortex shortly.


Place the samples on a Vortex adapter (e.g. Qiagen) and vortex at maximum speed for Duration00:10:00 .

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 Duration00:30:00 at Temperature65 °C and at maximum RPM.



10m
Centrifigation2500 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 Amount5 mL ammonium acetate buffer , vortex shortly, and incubate at Temperature4 °C for Duration00:10:00 .

10m
Centrifigation2500 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 Amount4 mL of inhibitor removal buffer . A precipitate may form. Vortex shortly, incubate at Temperature4 °C for Duration00:10:00 .


10m
Centrifigation2500 x g, 20°C, 00:04:00 . The solution will clear up. Avoiding the pellet, transfer up to Amount15 mL to a new tube.


4m
Add Amount30 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.


Centrifigation2500 x g, 20°C, 00:00:30 . Discard the flow-through. Repeat once to bind the complete sample volume.

30s
Add Amount10 mL wash buffer . Centrifigation2500 x g, 20°C, 00:05:00 to wash and dry the column.

5m
Transfer the column to a new tube. Add Amount1 mL elution buffer . Incubate for Duration00:03:00 at TemperatureRoom temperature .

3m
Centrifigation2500 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 250 mg of input material
Protocol for up to 250 mg of input material
50m
50m
Prepare one 2 mL centrifuge tube per sample with 750 mg of garnet beads.
Add Amount250 mg of soil or sediment sample.

Add Amount750 µL bead-beating solution and Amount60 µL lysis solution . Vortex shortly.

Place the samples on a Vortex adapter (e.g. Qiagen) and vortex at maximum speed for Duration00:10:00 .

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 Duration00:30:00 at Temperature65 °C and at maximum RPM.

10m
Centrifigation10000 x g, 20°C, 00:03:00 . Transfer the supernatant to a new tube.

3m
Add Amount250 µL ammonium acetate buffer , vortex shortly, and incubate at Temperature4 °C for Duration00:05:00 .
5m
Centrifigation10000 x g, 20°C, 00:01:00 . Transfer the supernatant to a new tube.

1m
Add Amount200 µL of inhibitor removal buffer . A precipitate may form. Vortex shortly, incubate at Temperature4 °C for Duration00:05:00 .
5m
Centrifigation10000 x g, 20°C, 00:01:00 . Transfer Amount600 µL of the supernatant to a new tube.

1m
Add Amount1200 µL DNA binding buffer . Vortex to mix.

Load Amount650 µL of the mixture to a mini spin column (e.g. Epoch Life Science).

Centrifigation10000 x g, 20°C, 00:00:30 . Discard the flow-through. Repeat two times to bind the complete sample volume.

30s
Add Amount500 µL wash buffer . Centrifigation10000 x g, 20°C, 00:00:30 to wash the column. Discard the flow-through.
30s
Centrifigation10000 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 Amount50 µL elution buffer . Incubate for Duration00:03:00 at TemperatureRoom temperature .

3m
Centrifigation10000 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:
https://www.nature.com/articles/s41587-020-0588-y