Jan 24, 2025
Inhibitor-free DNA extraction from unsorted bulk samples
- Dominik Buchner1,
- Marie Borowski1
- 1University of Duisburg-Essen, Aquatic Ecosystem Research
- Aquatic Ecosystem Research
Protocol Citation: Dominik Buchner, Marie Borowski 2025. Inhibitor-free DNA extraction from unsorted bulk samples. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk96d5v5r/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: January 14, 2025
Last Modified: January 24, 2025
Protocol Integer ID: 118297
Keywords: dna, extraction, inhibitor removal, unsorted bulk sample
Abstract
This protocol describes how to extract inhibitor-free DNA from unsorted, homogenized bulk samples. 20 mL of homogenate can be processed in one extraction, but there is also a miniaturized version for 1 mL 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
Acetic acid Acetic acidCarl RothCatalog #7332.1
Labware:
50 mL centrifuge tubes, Ultra-High Performance Centrifuge tubes Ultra-High PerformanceVWR InternationalCatalog #525-1098
Garnet Sharp Particles Garnet Sharp ParticlesBioSpec ProductsCatalog #11079103gar
Zirconia Beads, 2 mm diameter Zirconia beads, 2 mm diaBioSpec ProductsCatalog #11079124zx
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
1.5 mL centrifuge tubes Reaction tube, 1.5 ml, PPSarstedtCatalog #72.690.001
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 Molarity (M)
- 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 Molarity (M) 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 L Tris stock solution 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
1 L Proteinase K storage buffer (50 millimolar (mM) Tris , 3 millimolar (mM) CaCl2 , 50 % (v/v) glycerol 7.8
- Add 50 mL of 1 Molarity (M) Tris stock solution 7.5
- Add 333 mg calcium chloride
- Add 500 mL of glycerol 87%
- Adjust volume to 900 mL with ddH2O
- Adjust pH to 7.8 with sodium hydroxide
- Adjust volume to 1 L 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 Molarity (M) 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 20 mL of input material
Protocol for up to 20 mL of input material
43m 30s
43m 30s
Prepare one 50 mL centrifuge tube per sample with 7.5 g of garnet beads (0.3 mm diameter) and 7.5 g of zirconia beads (2 mm diameter).
Add up to 20 mL bulk sample in ethanol to the tube.
Tube with up to 20 mL of bulk sample in ethanol
4000 x g, Room temperature, 00:03:00 to pellet the material. Decant as much ethanol as possible.
After centrifugation the material is pelleted
After decanting the ethanol.
3m
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 .
10m
1400 rpm, 56°C, 00:30:00 to fully lyse the material.
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 00:05:00 .
5m
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 00:05:00 .
5m
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 1 mL of input material
Protocol for up to 1 mL of input material
56m
56m
Prepare one 2 mL centrifuge tube per sample with 750 mg of garnet beads (0.3 mm diameter) and 750 mg of zirconia beads (2 mm diameter).
Add 1000 µL of unsorted bulk sample.
4000 x g, 20°C, 00:03:00 and remove as much of the ethanol as possible without disturbing the pellet.
3m
Add 1380 µL bead-beating solution , 240 µL lysis solution and 100 µL Proteinase K . Vortex shortly.
Place the samples in a bead-beater (e.g. BioSpec Products Mini Bead-beater) and bead-beat at maximum speed for 00:02:00 . If no bead-beater is available this step can be skipped, but the yield might decrease.
2m
Place the samples on a Thermonlock and incubate for 00:30:00 at 56 °C and at maximum RPM
30m
10000 x g, 20°C, 00:03:00 . Transfer 600 µL of the supernatant to a new tube.
Note
If you want to extract technical replicates, this is the right time to split your samples into 2x 300 µL .
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.
Note
Depending on the amount of lysate and / or technical replication the amount of binding buffer differs. General rule: The amount of binding buffer should be at least twice the amount of lysate / sample material.
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 .
Note
The amount of elution buffer is variable and can range from 30 to 200 µL depending if concentrated sample or more volume is required.
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