Jan 07, 2025

Inhibitor removal from DNA extracts

DOI
https://dx.doi.org/10.17504/protocols.io.rm7vzbp54vx1/v1
Inhibitor removal from DNA extracts
  • 1University of Duisburg-Essen, Aquatic Ecosystem Research
  • Aquatic Ecosystem Research
Dominik Buchner
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DOI: https://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
Keywords: inhibitor removal from dna, inhibitor removal, extracted dna, inhibitor, inihibitory substances such as humic substance, humic substance, dna, inihibitory substance, protocol
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 100 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 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
Acetic acid Acetic acidCarl RothCatalog #7332.1
Ethanol absolute Ethanol absolute 99.8% p.a.Carl RothCatalog #9065.1



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

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 , 0.05 % (v/v) Tween 20 , 120 millimolar (mM) sodium acetate ) 5
  • Add 119.4 g guanidine hydrochloride to a beaker
  • Fill up to 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









Before start
Make sure all buffers are prepared before starting.
Inhibitor removal from DNA extracts
31m
Prepare 100 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 100 µL . Differing volumes can be used, however, all the following volums have to be adjusted accordingly. For volumes smaller than 100 µL we recommend to simply fill up the sample to 100 µL with water and then proceed with the protocol. If a sample volume of 200 µL is to be processed, all volumes have to be doubled.


Add 345 µL bead-beating solution and 60 µL lysis solution .
Vortex shortly.
10m
10000 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
Add125 µL ammonium acetat 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 100 µL of inhibitor removal buffer . A precipitate may form. Vortex shortly, incubate at4 °C for00:05:00


5m
10000 x g, 20°C, 00:01:00 . Transfer600 µL of the supernatant to a new tube.

1m
Add1200 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 1.5mL microcentrifuge tube.

1m
Add50 µL elution buffer . Incubate for 00:03:00 atRoom temperature .

3m
10000 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