Jan 07, 2025

Public workspaceInhibitor removal from DNA extracts

DOI
dx.doi.org/10.17504/protocols.io.rm7vzbp54vx1/v1
  • 1University of Duisburg-Essen, Aquatic Ecosystem Research
  • Aquatic Ecosystem Research
Dominik Buchner
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzbp54vx1/v1
Protocol Citation: Dominik Buchner, Marie Borowski 2025. Inhibitor removal from DNA extracts. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzbp54vx1/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: April 21, 2023
Last Modified: January 07, 2025
Protocol Integer ID: 80902
Abstract
This protocol describes how to remove inihibitory substances such as humic substances from DNA that has already been extracted. The protocol is formulated for an initial input of Amount100 µL of extracted DNA however it can be adjusted to any other volume as well with minor changes. 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
Acetic acid ReagentAcetic acidCarl RothCatalog #7332.1
Ethanol absolute ReagentEthanol absolute 99.8% p.a.Carl RothCatalog #9065.1



Labware:
2 mL centrifuge tubes ReagentReaction tube, 2 mL, PPSarstedtCatalog #72.691
1.5 mL centrifuge tubes ReagentReaction tube, 1.5 ml, PPSarstedtCatalog #72.690.001
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

Amount1 L Tris stock solution 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 (Concentration2.5 Molarity (M) Guanidine hydrochloride , Concentration80 % (v/v) ethanol , Concentration0.05 % (v/v) Tween 20 , Concentration120 millimolar (mM) sodium acetate ) Ph5
  • Add Amount119.4 g guanidine hydrochloride to a beaker
  • Fill up to Amount400 mL ethanol
  • 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









Before start
Make sure all buffers are prepared before starting.
Inhibitor removal from DNA extracts
Inhibitor removal from DNA extracts
31m
31m
Prepare Amount100 µL of sample in 2 mL tubes.

Sample contaminated with inhibitory substance (e.g. humic acids)

Note
This protocol is designed for a sample volume of Amount100 µL . Differing volumes can be used, however, all the following volums have to be adjusted accordingly. For volumes smaller than Amount100 µL we recommend to simply fill up the sample to Amount100 µL with water and then proceed with the protocol. If a sample volume of Amount200 µL is to be processed, all volumes have to be doubled.


Add Amount345 µL bead-beating solution and Amount60 µL lysis solution .
Vortex shortly.
10m
Centrifigation10000 x g, 20°C , 00:03:00 . Transfer all of the supernatant to a new tube.

Try to avoid the the pellet if any is formed.

3m
AddAmount125 µL ammonium acetat 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 Amount100 µL of inhibitor removal buffer . A precipitate may form. Vortex shortly, incubate atTemperature4 °C forDuration00:05:00


5m
Centrifigation10000 x g, 20°C, 00:01:00 . TransferAmount600 µL of the supernatant to a new tube.

1m
AddAmount1200 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 1.5mL microcentrifuge tube.

1m
AddAmount50 µL elution buffer . Incubate for Duration00:03:00 atTemperatureRoom temperature .

3m
Centrifigation10000 x g, 20°C, 00:01:00 to eluate the DNA. DNA eluate should be completely colorless and ready to go for downstrom analysis.




1m