Soil quality - Method to directly extract DNA from soil samples (ISO 11063:2012)

This International Standard specifies a method for direct extraction of DNA from soil samples to analyse the
global structure and the abundance of soil bacterial communities using PCR-based technologies. This method
is mainly dedicated to agricultural and forest soils. This method can possibly not be suitable for soils rich in
organic matter (e.g. peat soils) or soils heavily polluted with organic pollutants or heavy metals.
The direct extraction of DNA from soil samples provides unique insight into the richness and structure of
microbial communities which are key parameters to estimate the biodiversity of soil microbiota. Molecular
approaches based on PCR (polymerase chain reaction) amplification of soil DNA constitute a promising
domain and can contribute in the near future to the development of routine tools to monitor the microbiota of
soil environments.
Users of the method ought to be aware that although soil submitted to the DNA extraction procedure is sieved
thoroughly (2 mm mesh, procedure described in 5.1), plant residues can still remain in soil samples and, as a
result, traces of plant DNA can contaminate the soil DNA extract.

Bodenbeschaffenheit - Verfahren zur direkten Extraktion von DNA aus Bodenproben (ISO 11063:2012)

Diese Norm legt ein Verfahren zur direkten Extraktion von DNA aus Bodenproben für die Analyse der allgemeinen Struktur und der Abundanz von Lebensgemeinschaften von Bodenbakterien mit Verfahrensweisen auf der Grundlage von PCR fest. Dieses Verfahren ist vorwiegend für landwirtschaftlich genutzte Böden und Waldboden vorgesehen. Dieses Verfahren kann möglicherweise für Böden mit hohem organischen Anteil (z. B. Torfböden) oder Böden, die stark mit organischen Verunreinigungen oder Schwermetallen belastet sind, geeignet sein.
Die direkte Extraktion von DNA aus Bodenproben bietet einen einzigartigen Einblick in den Artenreichtum und die Struktur mikrobieller Lebensgemeinschaften, was die Schlüsselparameter zur Abschätzung der Biodiver-sität der Bodenmikroflora und -fauna sind. Molekulare Verfahren auf der Grundlage der Amplifikation von Boden DNA durch PCR (Polymerasekettenreaktion) stellen ein vielversprechendes Gebiet dar und können in naher Zukunft zur Entwicklung von Routinewerkzeugen beitragen, die die Abschätzung der Bodenbeschaf-fenheit ermöglichen.
Anwender dieser Norm sollten beachten, dass obwohl der Boden für die DNA-Extraktion sorgfältig gesiebt wird (2-mm-Sieb, Verfahren in 5.1 beschrieben), Pflanzenrückstände in den Bodenproben noch verbleiben können und damit Spuren von Pflanzen-DNA das Boden-Extrakt verunreinigen können.

Qualité du sol - Méthode pour extraire directement l'ADN d'échantillons de sol (ISO 11063:2012)

La présente Norme internationale spécifie une méthode pour extraire directement l'ADN d'échantillons de
sol en vue d'analyser la structure globale et l'abondance des communautés microbiennes du sol en utilisant
des techniques de PCR. Cette méthode est principalement destinée aux sols agricoles et forestiers. Cette
méthode peut ne pas être appropriée aux sols riches en matières organiques (par exemple sols de tourbières)
ou aux sols très pollués par des polluants organiques ou des métaux lourds.
L'extraction directe de l'ADN d'échantillons de sol fournit des informations précieuses sur l'abondance et
la structure des communautés microbiennes qui sont des paramètres clés pour estimer la biodiversité des
communautés microbiennes telluriques. Les approches moléculaires utilisant l'amplification PCR (amplification
en chaîne par polymérisation) de l'ADN extrait du sol offrent des perspectives prometteuses et peuvent
contribuer, dans un futur proche, au développement d'outils de routine permettant de surveiller la qualité de la
composante microbienne des sols.
L'utilisateur de la méthode doit savoir que, même si le sol soumis au mode opératoire d'extraction de l'ADN
est soigneusement tamisé (mailles de 2 mm, mode opératoire décrit en 5.1), des résidus végétaux peuvent
demeurer dans les échantillons de sol et de ce fait, l'extrait d'ADN prélevé du sol peut être contaminé par des
traces d'ADN végétal.

Kakovost tal - Metoda neposredne ekstrakcije DNK iz vzorcev tal (ISO 11063:2012)

Ta mednarodni standard določa metodo neposredne ekstrakcije DNK iz vzorcev tal za analizo globalne strukture in številčnosti bakterijskih skupnosti v tleh z uporabo tehnologij na podlagi polimerazne verižne reakcije (PCR). Ta metoda se uporablja predvsem za kmetijska in gozdna tla. Ta metoda je lahko neprimerna za tla, bogata z organsko snovjo (npr. šotna tla), ali tla, ki so močno onesnažena z organskimi onesnaževali ali težkimi kovinami. Neposredna ekstrakcija DNK iz vzorcev tal omogoča enkraten vpogled v bogatost in strukturo mikrobnih skupnosti, ki so ključni parametri za oceno biotske raznovrstnosti talne mikrobiote. Molekularni pristopi, ki temeljijo na okrepitvi PCR (polimerazne verižne reakcije) DNK v tleh, predstavljajo obetavno domeno in lahko v bližnji prihodnosti prispevajo k razvoju rutinskih orodij za spremljanje mikrobiote talnih okolij. Uporabniki metode se morajo zavedati, da lahko kljub temeljitemu presejanju tal v postopku ekstrakcije DNK (z 2 mm mrežo, postopek je opisan v točki 5.1), v vzorcih tal še vedno ostanejo ostanki rastlin, zato lahko sledovi rastlinskega DNK onesnažijo ekstrakt DNK tal.

General Information

Status
Withdrawn
Publication Date
19-Feb-2013
Current Stage
9960 - Withdrawal effective - Withdrawal
Completion Date
14-Oct-2020

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SLOVENSKI STANDARD
SIST EN ISO 11063:2013
01-maj-2013
Kakovost tal - Metoda neposredne ekstrakcije DNK iz vzorcev tal (ISO 11063:2012)
Soil quality - Method to directly extract DNA from soil samples (ISO 11063:2012)
Bodenbeschaffenheit - Verfahren zur direkten Extraktion von DNA aus Bodenproben
(ISO 11063:2012)

Qualité du sol - Méthode pour extraire directement l'ADN d'échantillons de sol (ISO

11063:2012)
Ta slovenski standard je istoveten z: EN ISO 11063:2013
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 11063:2013 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 11063:2013
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SIST EN ISO 11063:2013
EUROPEAN STANDARD
EN ISO 11063
NORME EUROPÉENNE
EUROPÄISCHE NORM
February 2013
ICS 13.080.30
English Version
Soil quality - Method to directly extract DNA from soil samples
(ISO 11063:2012)

Qualité du sol - Méthode pour extraire directement l'ADN Bodenbeschaffenheit - Verfahren zur direkten Extraktion

d'échantillons de sol (ISO 11063:2012) von DNA aus Bodenproben (ISO 11063:2012)
This European Standard was approved by CEN on 5 February 2013.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European

Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national

standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by translation

under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same

status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United

Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels

© 2013 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11063:2013: E

worldwide for CEN national Members.
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SIST EN ISO 11063:2013
EN ISO 11063:2013 (E)
Contents Page

Foreword ....................................................................................................................................................... 3

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SIST EN ISO 11063:2013
EN ISO 11063:2013 (E)
Foreword

The text of ISO 11063:2012 has been prepared by Technical Committee ISO/TC 190 “Soil quality” of the

International Organization for Standardization (ISO) and has been taken over as EN ISO 11063:2013 by

Technical Committee CEN/TC 345 “Characterization of soils” the secretariat of which is held by NEN.

This European Standard shall be given the status of a national standard, either by publication of an identical

text or by endorsement, at the latest by August 2013, and conflicting national standards shall be withdrawn at

the latest by August 2013.

Attention is drawn to the possibility that some of the elements of this document may be the subject of patent

rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.

According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following

countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech

Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece,

Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,

Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.

Endorsement notice

The text of ISO 11063:2012 has been approved by CEN as EN ISO 11063:2013 without any modification.

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SIST EN ISO 11063:2013
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SIST EN ISO 11063:2013
INTERNATIONAL ISO
STANDARD 11063
First edition
2012-02-01
Soil quality — Method to directly extract
DNA from soil samples
Qualité du sol — Méthode pour extraire directement l’ADN
d’échantillons de sol
Reference number
ISO 11063:2012(E)
ISO 2012
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SIST EN ISO 11063:2013
ISO 11063:2012(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2012

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,

electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO’s

member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2012 – All rights reserved
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SIST EN ISO 11063:2013
ISO 11063:2012(E)
Contents Page

Foreword ............................................................................................................................................................................iv

Introduction ........................................................................................................................................................................ v

1 Scope ...................................................................................................................................................................... 1

2 Normative references ......................................................................................................................................... 1

3 Terms and definitions ......................................................................................................................................... 1

4 Principle ................................................................................................................................................................. 1

5 Test materials ....................................................................................................................................................... 2

5.1 Soil ........................................................................................................................................................................... 2

5.2 Chemicals .............................................................................................................................................................. 2

5.3 Buffers and reagents .......................................................................................................................................... 3

6 Apparatus .............................................................................................................................................................. 4

7 Procedures ............................................................................................................................................................ 4

7.1 Preparation of soil samples .............................................................................................................................. 4

7.2 Mechanical and chemical lyses ....................................................................................................................... 4

7.3 Protein precipitation ........................................................................................................................................... 4

7.4 Nucleic acid precipitation and washing ........................................................................................................ 4

7.5 Nucleic acid storage ........................................................................................................................................... 4

8 Estimation of soil DNA quality and quantity ................................................................................................ 5

8.1 Soil DNA quality and purity............................................................................................................................... 5

8.2 Soil DNA quantity ................................................................................................................................................ 5

9 Validation of the extraction procedure .......................................................................................................... 5

10 International ring test ......................................................................................................................................... 5

11 Test report ............................................................................................................................................................. 5

Annex A (informative) International ring test for evaluating soil DNA extraction procedure ........................ 7

Bibliography .....................................................................................................................................................................21

© ISO 2012 – All rights reserved iii
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SIST EN ISO 11063:2013
ISO 11063:2012(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies

(ISO member bodies). The work of preparing International Standards is normally carried out through ISO

technical committees. Each member body interested in a subject for which a technical committee has been

established has the right to be represented on that committee. International organizations, governmental and

non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International

Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.

International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.

The main task of technical committees is to prepare International Standards. Draft International Standards

adopted by the technical committees are circulated to the member bodies for voting. Publication as an

International Standard requires approval by at least 75 % of the member bodies casting a vote.

Attention is drawn to the possibility that some of the elements of this document may be the subject of patent

rights. ISO shall not be held responsible for identifying any or all such patent rights.

ISO 11063 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological methods.

iv © ISO 2012 – All rights reserved
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SIST EN ISO 11063:2013
ISO 11063:2012(E)
Introduction

DNA (deoxyribonucleic acid) is an essential component of any living organism coding for enzymes responsible

for any biological activities. The study of DNA sequences from DNA sources extracted from different matrixes,

by means of numerous molecular approaches, provides molecular markers that can be used to sharply

distinguish and identify different organisms (bacteria, archaea and eucaryotes).

Up to now, most of the studies aiming to develop microbial soil quality indicators applicable to complex

environments, such as soil, were biased by the unculturability of many microorganisms and the lack of sensitivity

[16]

of traditional microbiological methods . The recent development of numerous molecular biology methods

based primarily on amplification of soil-extracted nucleic acids have provided a pertinent alternative to classical

culture-based microbiological methods, providing unique insight into the composition, richness, and structure

[15], [18], [26], [27], [36]

of microbial communities . DNA-based approaches are now well-established in soil ecology

and serve as genotypic (= molecular genetic) markers for determining microbial diversity.

The results of molecular analyses of soil microbial communities and/or populations rely on two main parameters:

a) the extraction of DNA representative of the indigenous bacterial community composition;

b) PCR bias, such as the choice of primers, the concentration of amplified DNA, errors in the PCR, or

[23], [26], [38], [40]

even the method chosen for analysis . Recently, numerous studies have investigated new

[20]

methods to improve extraction, purification, amplification, and quantification of DNA from soils .

The aim of this International Standard is to describe the procedure used to extract DNA directly from soil

samples. The reproducibility of this soil DNA extraction procedure was assessed in an international ring-test

study (Annex A). The reproducibility of this soil DNA extraction procedure was successfully evaluated on both

quantitative (q-PCR) and qualitative (A-RISA) approaches.
© ISO 2012 – All rights reserved v
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SIST EN ISO 11063:2013
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SIST EN ISO 11063:2013
INTERNATIONAL STANDARD ISO 11063:2012(E)
Soil quality — Method to directly extract DNA from soil samples
1 Scope

This International Standard specifies a method for direct extraction of DNA from soil samples to analyse the

global structure and the abundance of soil bacterial communities using PCR-based technologies. This method

is mainly dedicated to agricultural and forest soils. This method can possibly not be suitable for soils rich in

organic matter (e.g. peat soils) or soils heavily polluted with organic pollutants or heavy metals.

The direct extraction of DNA from soil samples provides unique insight into the richness and structure of

microbial communities which are key parameters to estimate the biodiversity of soil microbiota. Molecular

approaches based on PCR (polymerase chain reaction) amplification of soil DNA constitute a promising

domain and can contribute in the near future to the development of routine tools to monitor the microbiota of

soil environments.

Users of the method ought to be aware that although soil submitted to the DNA extraction procedure is sieved

thoroughly (2 mm mesh, procedure described in 5.1), plant residues can still remain in soil samples and, as a

result, traces of plant DNA can contaminate the soil DNA extract.
2 Normative references

The following referenced documents are indispensable for the application of this document. For dated

references, only the edition cited applies. For undated references, the latest edition of the referenced document

(including any amendments) applies.

ISO 10381-6, Soil quality — Sampling — Part 6: Guidance on the collection, handling and storage of soil under

aerobic conditions for the assessment of microbiological processes, biomass and diversity in the laboratory

3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
soil DNA

DNA extracted from soil-living microorganisms and remaining DNA from dead microorganisms

4 Principle

DNA is directly extracted from 0,25 g soil samples using the following extraction procedure. This method

reliably allowed analysing the global structure of bacterial and archeal communities and could be adapted

[32]

(extraction from a 1 g soil sample) to assess the global structure of fungal communities . Soil samples added

with an extraction buffer are submitted to mechanical and chemical lyses. The lysis step, e.g. by bead beating,

is a crucial step to also extract DNA from microbes that are difficult to lyse. After a brief centrifugation, soil

debris are removed and proteins are precipitated with potassium acetate. After centrifugation, the supernatant

is recovered and nucleic acids are precipitated with ice-cold isopropanol. After centrifugation, the nucleic acids

pellet is washed with 70 % ethanol and suspended in sterile ultra-pure water. DNA quality is then checked by

electrophoresis on an agarose gel and the DNA quantity is estimated using a spectro-fluorimeter. A schematic

overview of the procedure is given in Figure 1.
© ISO 2012 – All rights reserved 1
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SIST EN ISO 11063:2013
ISO 11063:2012(E)
5 Test materials
5.1 Soil

Soil samples should be collected and sieved (2 mm mesh). If samples are not immediately processed, they

should be stored for up to two years at −20 °C or up to 10 years at −80 °C or in liquid nitrogen (−180 °C) as

specified in ISO 10381-6. If soil samples are frozen, they may be thawed only once. Some of these storage

conditions are currently under testing.
Figure 1 — Schematic overview of soil DNA extraction procedure
5.2 Chemicals
5.2.1 Tris[hydroxymethyl]aminomethane, C H NO (CAS No. 77-86-1).
4 11 3

5.2.2 Ethylenediaminetetraacetic acid disodium salt (EDTA), C H N O Na ·2 H O (CAS No. 6381-92 6).

10 14 2 8 2 2
5.2.3 Sodium chloride, NaCl (CAS No. 7647-14-5).
2 © ISO 2012 – All rights reserved
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SIST EN ISO 11063:2013
ISO 11063:2012(E)
5.2.4 Sodium dodecyl sulfate (SDS), CH (CH ) OSO Na (CAS No. 151-21-3).
3 2 11 3
5.2.5 Polyvinylpyrrolidone (PVP), [C H NO] (CAS No. 9003-39-8).
6 9 n
5.2.6 Sodium acetate, CH COONa (CAS No. 6131-90-4).
5.2.7 Acetic acid or glacial acetic acid, CH COOH (CAS No. 64-19-7).
5.2.8. Isopropanol, CH CHOHCH (CAS No. 67-63-0).
3 3
5.2.9 Ethanol, CH CH OH (CAS No. 64-17-5).
3 2
5.2.10 Molecular-biology-grade water, H O.
5.3 Buffers and reagents

Buffers and reagents (except intercalent molecules) used for soil DNA extraction are sterilized (120 °C for

20 min) and stored at room temperature. Ethanol and isopropanol are stored at −20 °C.

5.3.1 Tris-HCl, 1 mol/l, 121,14 g of tris in 1 000 ml of H O, adjusting with 4 mol/l HCl to pH 8,0.

5.3.2 EDTA, 0,5 mol/l, 186,10 g of EDTA in 1 000 ml of H O, adjusting with NaOH (10 mol/l) to pH 8,0.

5.3.3 NaCl, 1 mol/l, 58,44 g of NaCl in 1 000 ml of H O.
5.3.4 PVP 40, 20 %, 200 g of PVP in 1 000 ml of H O.
5.3.5 SDS, 20 %, 200 g of SDS in 1 000 ml of H O.

5.3.6 Homogenization buffer (newly prepared just before being used), 100 ml of 1 mol/l tris-HCl (pH 8,0), 200 ml

of 0,5 mol/l EDTA (pH 8,0), 100 ml of 1 mol/l NaCl, 50 ml of 20 % PVP 40, 100 ml of 20 % SDS in 450 ml of H O.

5.3.7 Sodium acetate, 5 mol/l (pH 5,5), 410,15 g of CH COONa in 800 ml of H O. Add 120 ml of acetic acid

3 2

and then adjust the pH to 5,5 with glacial acetic acid. Add water to make up to 1 000 ml.

5.3.8 Ethanol, 70 %, 700 ml of pure ethanol in 300 ml of H O.
5.3.9 TE buffer, pH 8,0, 10 mmol/l tris-HCl, 1 mmol/l EDTA.
5.3.10 Glass beads (106 µm).
5.3.11 Glass beads (2 mm).
5.3.12 Ethidium bromide, 5 mg of ethidium bromide in 1 000 ml of H O.

5.3.13 Fluorescent nucleic acid stain, excitation at 480 nm and emission at 520 nm.

5.3.14 Pure DNA (100 ng/µl)

5.3.15 TBE buffer × 10, pH 8,0, 108 g of tris base, 55 g of boric acid, 40 ml of 0,5 mol/l EDTA (pH 8,0) in

1 000 ml of H O.
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SIST EN ISO 11063:2013
ISO 11063:2012(E)
5.3.16 TBE buffer × 1, 100 ml of TBE buffer × 10 in 900 ml of H O.
6 Apparatus

Use standard laboratory equipment including pipettes, a centrifuge, fume hood cabinet, horizontal

electrophoresis system and the following.

6.1 Mini-bead beating apparatus, with a beating frequency varying from, for example, 100 min to

2 600 min and a 16 mm amplitude of agitation.

6.2 Spectro-fluorimeter, allowing the quantification of double-strand DNA at 520 nm with a fluorescent

nucleic acid stain excited at 480 nm.
7 Procedures
7.1 Preparation of soil samples

Weigh 0,25 g of soil (equivalent dry mass) in 2 ml micro-tubes just before extracting, or immediately freeze the

soil sample in liquid nitrogen and keep it frozen at −80 °C until its use.
7.2 Mechanical and chemical lyses

Add 0,5 g of 106 µm glass beads (wear a mask for protection) and two glass beads (2 mm diameter) to the soil

sample. Add 1 ml of homogenization buffer (composition given in 5.3.6). Agitate the soil samples 1 600g for

30 s (16 mm of amplitude) using a bead-beating system (tube support previously placed at −20 °C). Incubate

at 70 °C for 10 min. Centrifuge for 1 min at 14 000g (4 °C). Carefully recover the supernatant and transfer it to

a new 2 ml microtube.
7.3 Protein precipitation

To the supernatant obtained in 7.2, add 5 mol/l sodium acetate (pH 5,5) (composition given in 5.3.7) of an

amount that is 1/10 of the volume of the supernatant. Mix by vortexing and incubate on ice for 10 min. Centrifuge

for 5 min at 14 000g (4 °C). Carefully recover the supernatant and transfer it to a new 1,5 ml microtube.

7.4 Nucleic acid precipitation and washing

Perform all these steps below a fume hood because of dangerous isopropanol vapours. Liquid and solid

wastes shall be evacuated as chemical waste.

To the supernatant obtained in 7.3, add cold isopropanol (-20 °C) of an amount that is 1/1 of the volume of the

supernatant. Incubate the samples at −20 °C for 15 min. Centrifuge for 30 min at 14 000g (4 °C). Carefully

eliminate the supernatant. Wash the nucleic acids pellet with cold 70 % ethanol (do not resuspend the pellet).

Centrifuge for 15 min at 14 000g (4 °C). Eliminate any traces of ethanol and let the nucleic acid pellet dry for

15 min at 37 °C. Suspend the pellet in 100 µl of ultra-pure water or TE buffer (pH 8) (composition given in 5.3.9).

7.5 Nucleic acid storage

Aliquot the soil DNA (4 × 25 µl) and store the DNA samples at −20 °C until their use. Repeated freezing and

thawing of the DNA extracts should be omitted.
4 © ISO 2012 – All rights reserved
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SIST EN ISO 11063:2013
ISO 11063:2012(E)
8 Estimation of soil DNA quality and quantity
8.1 Soil DNA quality and purity

The quality and the size of the soil DNA are checked by electrophoresis on 1 % agarose gels in TBE buffer. Gels

are stained with appropriate staining (e.g. ethidium bromide, 5 mg/l). The purity of the soil DNA is assessed by

[11]

spectrophotometry at 260 nm for the DNA analysis and at 400 nm for humic acid substances .

The step of chemical and mechanical lysis is critical, and it should be adequate to lyse a representative portion

[39]
of microbes but avoid fragmentation of the DNA .
DNA extracts which are still slightly brownish need a further DNA purification.
8.2 Soil DNA quantity

The soil DNA content is determined using a fluorescent nucleic acid stain (5.3.13) which fluoresces when

intercalated within the double helix of DNA. A calibration curve relating the amount of standard DNA (5 ng,

10 ng, 20 ng, 50 ng, 100 ng, 150 ng and 200 ng of pure DNA) to the amount of fluorescence quantified is

established and used to estimate the amount of DNA extracted from the soil. Measurements are performed

using a spectro-fluorimeter (6.2). The analysis is carried out by relevant software.

Alternatively, the soil DNA content can be determined by resolving soil DNA extracts by electrophoresis in a

1 % agarose gel, stained with ethidium bromide and photographed under a camera. Dilutions of pure DNA

were included in each gel and a standard curve of DNA concentration (1 000 ng, 500 ng, 250 ng, 125 ng,

62,5 ng to 31,25 ng). The ethidium bromide intensity was integrated to establish a standard curve used for

estimating soil DNA concentration as described previously by Reference [32].

Alternatively, the soil DNA content can be determined by spectrophotometry at 260 nm when soil DNA is lowly

contaminated with humic acid substances (400 nm) and proteins (A260/A280 averaging 1,6).

9 Validation of the extraction procedure

The laboratory can validate the procedure of soil DNA extraction by processing the reference soil and comparing

the obtained yield of soil DNA extraction to the expected one.
10 International ring test

This method for extracting soil DNA was evaluated through an international ring test involving nine different

laboratories working on six different European soils. The report of this ring test is provided in Annex A.

11 Test report
The test report shall include the following information:
a) a reference to this International Standard: ISO 11063:2012;
b) soil collection, including date and place (GPS coordinates) of collection;

c) treatment and storage of soil sample (e.g. sieving method, conditions and length of storage);

d) physical and chemical characteristics of the soil;
e) quantity of soil used for DNA extraction;
f) date(s) of extraction;
g) duration of nucleic acids storage (if appropriate);
© ISO 2012 – All rights reserved 5
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SIST EN ISO 11063:2013
ISO 11063:2012(E)

h) tables of results including concentration of soil DNA extracts and amount of DNA extracted per gram of

soil (dry weight equivalent);

i) any details not specified in this International Standard or which are optional, as well as any effect which

may have affected the results.
6 © ISO 2012 – All rights reserved
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SIST EN ISO 11063:2013
ISO 11063:2012(E)
Annex A
(informative)
International ring test for evaluating soil DNA extraction procedure
A.1 Introduction

Up to now, most of the microbial diversity studies conducted in complex ecosystems, such as soil, has been

biased essentially by the unculturability of many microorganisms and the lack of sensitivity of traditional

microbiological methods. In the past decade, applications of numerous molecular biology methods based

primarily on amplification of indirect or direct soil-extracted nucleic acids (DNA or DNA/RNA) have provided

a pertinent alternative to classical culture-based microbiological methods, providing unique insight into the

[15], [18], [26], [27], [36]

composition, richness and structure of microbial communities . DNA-based approaches

are now well-established in soil ecology and serve as genotypic (= molecular genetic) markers for determining

microbial diversity.

However, the results of molecular analysis of microbial communities rely mostly on two main parameters:

a) the extraction of DNAs representative of the indigenous bacterial community composition;

b) PCR bias, such as the choice of primers, the concentration of amplified DNA, errors in the PCR, or even

the method chosen for analysis. Recently, numerous studies have investigated new methods to improve

extraction, purification, amplification, and quantification of DNA from soils.

Although comparative studies have been performed to analyse the efficiency of methods for extraction and

purification of soil DNA recovered, the reproducibility of soil DNA extraction within different laboratories has not

yet been assessed. Therefore, in this context, the aim of this study was to evaluate the reproducibility of a soil

DNA extraction method developed by Reference [20].
A.2 Materials and methods
A.2.1 Laboratories involved in the study

Nine different laboratories from six European countries (France, Finland, Germany, Spain, Italy and Sweden)

were involved in the international ring test with the following organization. Laboratory in charge of the program:

INRA/Université de Bourgogne, Laboratoire de Microbiologie du Sol et de l’Environnement, (Dijon, France).

The participant laboratories are listed below: Institut National de l’Environnement Industriel et des Risques,

INERIS (Verneuil-en-Halatte, France); IPL santé, environnement durables Est, Laboratoire Etudes et Expertises

(Nancy, France); Swedish University of Agricultural Sciences (Uppsala, Sweden); GSF Munich (Munich,

Germany); Julius Kühn-Institut Bundesforschungsinsinstitut für Kulturpflanzen, JKI (Brauschweig, Germany);

Universita di Catania, DACPA-Sezione Scienze Agrochimiche (Catania, Italy); CSIC, Estacion Experimental

del Zaidin (Grenada, Spain); University of Helsinki (Helsinki, Finland).

A working group constituted of each responsible scientist of each research laboratory was established. The

main objectives of the working group were to:
— design the set-up of the inter-laboratories assay,
— analyse and discuss the results produced in this study.

The procedure used for the international ring test study is described below. The working group decided that

this assay should only concern the evaluation of the reproducibility of soil DNA extraction. To do so, the

working group decided that each laboratory should extract DNA from different soil samples following the same

protocol. Further work (DNA purification and DNA analyses) was only done by the laboratory leading this

© ISO 2012 – All rights reserved 7
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SIST EN ISO 11063:2013
ISO 11063:2012(E)

project (Laboratoire de Microbiologie du Sol et de l’Environnement, Dijon, France) in order to avoid adding

several biases due to these two additional steps.

Therefore, after soil DNA extraction, each laboratory involved in this project sent their samples to “Laboratoire

de Microbiologie du Sol et de l’Environnement” for further analyses. Upon their arrival, soil DNA samples were

purified as d
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