Soil quality - Estimation of abundance of selected microbial gene sequences by quantitative PCR from DNA directly extracted from soil (ISO 17601:2016)

ISO 17601:2016 specifies the crucial steps of a quantitative real-time polymerase chain reaction (qPCR) method to measure the abundance of selected microbial gene sequences from soil DNA extract which provides an estimation of selected microbial groups.
It is noteworthy that the number of genes is not necessarily directly linked to the number of organisms that are measured. For example, the number of ribosomal operon is ranging from one copy to 20 copies in different bacterial phyla. Therefore, the number of 16S rRNA sequences quantified from soil DNA extracts does not give an exact estimate of the number of soil bacteria. Furthermore, the number of sequences is not necessarily linked to living microorganisms and can comprise sequences amplified from dead microorganisms.

Bodenbeschaffenheit - Ermittlung der Häufigkeit ausgewählter mikrobieller Gensequenzen durch quantitative PCR aus DNA-Boden-Extrakten (ISO 17601:2016)

Diese Internationale Norm legt die entscheidenden Schritte eines Verfahrens der quantitativen Real-time-Polymerase-Kettenreaktion (qPCR) zur Messung der Häufigkeit ausgewählter mikrobieller Gensequenzen aus Boden-DNA-Extrakten fest, welche eine Quantifizierung ausgewählter mikrobieller Gruppen ermöglicht.
Dabei gilt, dass die Anzahl von Genen nicht zwangsläufig direkt mit der Anzahl an gemessenen Organismen korreliert. Zum Beispiel wurden für ribosomale Operons in unterschiedlichen Bakterienstämmen von einer bis zu 20 Kopien pro Zelle beschrieben. Daher ergibt die Quantifizierung von 16S rRNA-Sequenzen aus extrahierter DNA aus Bodenproben mittels qPCR nur einen Anhaltspunkt für die Häufigkeit von Bodenbakterien. Außerdem ist die DNA auch nach dem Zelltod in Böden persistent und daher kein Maß für die lebende Biomasse.

Qualité du sol - Estimation de l'abondance de séquences de gènes microbiens par amplification par réaction de polymérisation en chaîne (PCR) quantitative à partir d'ADN directement extrait du sol (ISO 17601:2016)

ISO 17601:2016 spécifie les étapes principales d'une méthode d'amplification par réaction de polymérisation en chaîne (PCR) quantitative (qPCR) permettant de mesurer l'abondance de séquences spécifiques de gènes microbiens à partir d'un extrait d'ADN du sol qui fournit une estimation de l'abondance de groupes microbiens spécifiques.
Il convient de noter que le nombre de gènes n'est pas nécessairement lié directement au nombre de micro-organismes mesurés. Par exemple, le nombre d'opérons ribosomiques est compris entre une et 20 copies dans différents phyla bactériens. Par conséquent, le nombre de séquences d'ARNr 16S quantifiées dans des extraits d'ADN du sol ne donne pas une estimation exacte du nombre de bactéries contenues dans le sol. Par ailleurs, le nombre de séquences n'est pas nécessairement lié à des micro-organismes vivants et peut comprendre des séquences amplifiées à partir de l'ADN extrait de micro-organismes morts.

Kakovost tal - Ocena številčnosti izbranih sekvenc mikrobnih genov s kvantitativnim PCR analizatorjem v talnih ekstraktih DNK (ISO 17601:2016)

Standard ISO 17601:2016 določa ključne korake pri metodi kvantitativne verižne reakcije s polimerazo v realnem času (qPCR) za merjenje številčnosti izbranih sekvenc mikrobnih genov v talnih ekstraktih DNK, ki zagotavlja oceno izbranih mikrobnih skupin.
Omeniti je treba, da število genov ni nujno neposredno povezano s številom organizmov, ki so izmerjeni. Število ribosomskih operonov lahko na primer znaša od ene do 20 kopij v različnih deblih bakterij. Zaradi tega število sekvenc 16S rRNA, kvantificiranih iz talnih ekstraktov DNK, ne zagotavlja točne ocene števila bakterij v tleh. Poleg tega število sekvenc ni nujni povezano z živimi mikroorganizmi in lahko zajema sekvence, povečane z mrtvimi mikroorganizmi.

General Information

Status
Published
Publication Date
20-Feb-2018
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
21-Feb-2018
Completion Date
21-Feb-2018

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SLOVENSKI STANDARD
SIST EN ISO 17601:2018
01-julij-2018
.DNRYRVWWDO2FHQDãWHYLOþQRVWLL]EUDQLKVHNYHQFPLNUREQLKJHQRYV
NYDQWLWDWLYQLP3&5DQDOL]DWRUMHPYWDOQLKHNVWUDNWLK'1. ,62
Soil quality - Estimation of abundance of selected microbial gene sequences by
quantitative PCR from DNA directly extracted from soil (ISO 17601:2016)
Bodenbeschaffenheit - Abschätzung der Häufigkeit ausgewählter mikrobieller
Gensequenzen durch quantitative PCR aus DNA-Boden-Extrakten (ISO 17601:2016)
Qualité du sol - Estimation de l'abondance de séquences de gènes microbiens par

amplification par réaction de polymérisation en chaîne (PCR) quantitative à partir d'ADN

directement extrait du sol (ISO 17601:2016)
Ta slovenski standard je istoveten z: EN ISO 17601:2018
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 17601:2018 en,fr,de

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

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SIST EN ISO 17601:2018
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SIST EN ISO 17601:2018
EN ISO 17601
EUROPEAN STANDARD
NORME EUROPÉENNE
February 2018
EUROPÄISCHE NORM
ICS 13.080.30
English Version
Soil quality - Estimation of abundance of selected
microbial gene sequences by quantitative PCR from DNA
directly extracted from soil (ISO 17601:2016)

Qualité du sol - Estimation de l'abondance de Bodenbeschaffenheit - Abschätzung der Häufigkeit

séquences de gènes microbiens par amplification par ausgewählter mikrobieller Gensequenzen durch

réaction de polymérisation en chaîne (PCR) quantitative PCR aus DNA-Boden-Extrakten (ISO

quantitative à partir d'ADN directement extrait du sol 17601:2016)
(ISO 17601:2016)
This European Standard was approved by CEN on 14 February 2018.

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, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 17601:2018 E

worldwide for CEN national Members.
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SIST EN ISO 17601:2018
EN ISO 17601:2018 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 17601:2018
EN ISO 17601:2018 (E)
European foreword

The text of ISO 17601:2016 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 17601:2018 by

Technical Committee CEN/TC 444 “Test methods for environmental characterization of solid matrices”

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 2018, and conflicting national standards shall

be withdrawn at the latest by August 2018.

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

patent rights. CEN 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, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and the United Kingdom.
Endorsement notice

The text of ISO 17601:2016 has been approved by CEN as EN ISO 17601:2018 without any modification.

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SIST EN ISO 17601:2018
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SIST EN ISO 17601:2018
INTERNATIONAL ISO
STANDARD 17601
First edition
2016-01-15
Soil quality — Estimation of
abundance of selected microbial gene
sequences by quantitative PCR from
DNA directly extracted from soil
Qualité du sol — Estimation de l’abondance de séquences de gènes
microbiens par amplification par réaction de polymérisation en
chaîne (PCR) quantitative à partir d’ADN directement extrait du sol
Reference number
ISO 17601:2016(E)
ISO 2016
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SIST EN ISO 17601:2018
ISO 17601:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland

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

or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior

written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of

the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved
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SIST EN ISO 17601:2018
ISO 17601:2016(E)
Contents Page

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

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

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

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

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

4 Principle ........................................................................................................................................................................................................................ 2

5 Test materials .......................................................................................................................................................................................................... 4

5.1 DNA ................................................................................................................................................................................................................... 4

5.2 Bacteria .......................................................................................................................................................................................................... 4

5.3 Plasmid .......................................................................................................................................................................................................... 4

5.4 Enzyme .......................................................................................................................................................................................................... 4

5.5 Chemicals ..................................................................................................................................................................................................... 4

5.6 Product for bacterial culture medium ................................................................................................................................ 5

5.7 Buffer and reagents ............................................................................................................................................................................. 5

6 Apparatus ..................................................................................................................................................................................................................... 6

7 Procedure..................................................................................................................................................................................................................... 6

7.1 qPCR standard preparation and calibration of qPCR assay (task 1) ........................................................ 6

7.1.1 General...................................................................................................................................................................................... 6

7.1.2 Amplicon design (task 1, step 1) ........................................................................................................................ 6

7.1.3 qPCR standard preparation (task 1, step 2) ............................................................................................. 7

7.1.4 Isolate DNA, environmental DNA, artificial DNA .................................................................................. 7

7.1.5 Calibration of the qPCR (task 1, step 3) ....................................................................................................... 9

7.2 Preparation of soil DNA template and inhibition test (task 2) ...................................................................10

7.2.1 General...................................................................................................................................................................................10

7.2.2 Soil DNA preparation (task 2, step 4) .........................................................................................................10

7.2.3 Inhibition test (task 2, step 5) ...........................................................................................................................10

7.2.4 Dilution of DNA template ......................................................................................................................................12

7.3 qPCR assay (task 3) ..........................................................................................................................................................................12

7.3.1 General...................................................................................................................................................................................12

7.3.2 qPCR ........................................................................................................................................................................................12

7.4 Validation and analysis of qPCR assay (task 4) ........................................................................................................12

7.4.1 General...................................................................................................................................................................................12

7.4.2 Validation of the qPCR assay ..............................................................................................................................12

7.4.3 Calculation of the copy number of the gene of interest in the soil DNA extract.....13

8 Examination of the critical steps of the qPCR assay ......................................................................................................14

9 Expression of the results of the qPCR assay ..........................................................................................................................14

10 International ring test .................................................................................................................................................................................14

11 Test report ................................................................................................................................................................................................................14

Annex A (informative) Description of principal steps of TaqMan qPCR assay ...................................................15

Annex B (informative) International ring-test for evaluating qPCR to quantify the

abundance of selected microbial gene sequences from DNA directly extracted from soil ..17

Bibliography .............................................................................................................................................................................................................................30

© ISO 2016 – All rights reserved iii
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SIST EN ISO 17601:2018
ISO 17601:2016(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.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).

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. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www.iso.org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation on the meaning of ISO specific terms and expressions related to conformity

assessment, as well as information about ISO’s adherence to the WTO principles in the Technical

Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information

The committee responsible for this document is ISO/TC 190, Soil quality, Subcommittee SC 4,

Biological methods.
iv © ISO 2016 – All rights reserved
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SIST EN ISO 17601:2018
ISO 17601:2016(E)
Introduction

DNA (DNAs) is a major component of any living organisms coding for enzymes responsible for

their biological activities. The study of DNA sequences from DNA sources extracted from different

environmental matrices, 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 quality indicators applicable to complex

environment such as soil were biased by the poor culturability of many microorganisms under

laboratory conditions and the lack of sensitivity of traditional microbiological methods. The recent

development of a large set of molecular biology methods based on amplification of soil-extracted

nucleic acids have provided a pertinent alternative to classical culture-based microbiological methods

[2]

providing unique insight into the composition, richness, and structure of microbial communities.

[3] [4] [5] [6]

DNA-based approaches are now well established in soil ecology and serve as genotypic

markers for determining microbial diversity. The results of molecular analyzes of soil microbial

communities and/or populations rely on two main parameters: a) the extraction of DNA representative

of the indigenous bacterial community composition and b) PCR bias such as the choice of primers, the

[7] [4] [8] [9]

concentration of amplified DNA, errors in the PCR, or even the method chosen for analysis.

Numerous studies have investigated new methods to improve extraction, purification, amplification,

[10]

and quantification of DNA from soils. Recently, ISO 11063 reporting “a method to extract nucleic

acids directly from soil samples“ derived from Reference [10] is opening a new window for developing

[11]
standardized molecular approaches to estimate soil quality.

The aim of this International Standard is to describe the procedure used to set up and perform

quantitative PCR to quantify the abundance of soil microbial phyla, as well as functional groups from

DNA directly extracted from soil samples. The quantification of soil microbial phyla, as well as functional

groups by qPCR assays can contribute to the development of routine tools to monitor soil quality.

The repeatability and the reproducibility of the procedure of the quantitative PCR were assessed in

an international ring test study (see Annex B). The repeatability of this procedure was successfully

evaluated for both 16S rRNA genes, as well as genes coding a functional marker of denitrifiers (the

nitrite reductase gene nirK). The reproducibility of this procedure revealed a laboratory effect which

can be overcome by interpreting the results of the quantification of the abundance of the microbial

groups by comparison, either by using an external reference (DNA extracted from a control strain) in

the assay or by calculating a percentage of variations between treatments to normalize the data.

© ISO 2016 – All rights reserved v
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SIST EN ISO 17601:2018
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SIST EN ISO 17601:2018
INTERNATIONAL STANDARD ISO 17601:2016(E)
Soil quality — Estimation of abundance of selected
microbial gene sequences by quantitative PCR from DNA
directly extracted from soil
1 Scope

This International Standard specifies the crucial steps of a quantitative real-time polymerase chain

reaction (qPCR) method to measure the abundance of selected microbial gene sequences from soil DNA

extract which provides an estimation of selected microbial groups.

It is noteworthy that the number of genes is not necessarily directly linked to the number of organisms

that are measured. For example, the number of ribosomal operon is ranging from one copy to 20 copies

in different bacterial phyla. Therefore, the number of 16S rRNA sequences quantified from soil DNA

extracts does not give an exact estimate of the number of soil bacteria. Furthermore, the number of

sequences is not necessarily linked to living microorganisms and can comprise sequences amplified

from dead microorganisms.
2 Normative references

The following documents, in whole or in part, are normatively referenced in this document and are

indispensable for its application. 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

ISO 11063, Soil quality — Method to directly extract DNA from soil samples
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
soil DNA
DNA extracted from soil of living and dead biota
EXAMPLE Microorganisms, plants, animals.
3.2
polymerase chain reaction
PCR

method allowing the amplification of a specific DNA sequence using a specific pair of oligonucleotide

primers
3.3
quantitative polymerase chain reaction
qPCR

method allowing the quantification in a DNA template (3.4) of the number of a specific DNA sequence

using a specific pair of oligonucleotide primers
3.4
template
DNA sample used to perform PCR (3.2) to amplify a specific DNA sequence
© ISO 2016 – All rights reserved 1
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SIST EN ISO 17601:2018
ISO 17601:2016(E)
3.5
amplicon
PCR product obtained by PCR (3.2) from a template (3.4)
3.6
cloning vector

circular DNA molecule in which the amplicon (3.5) is inserted by ligation used to transform competent

Escherichia coli for cloning the amplicon
3.7
qPCR standard

cloned DNA target used as template (3.4) for qPCR reaction to establish the standard curve relating the

abundance of target sequence as a function of cycle threshold values (Ct)
3.8
non-template control
NTC

control, usually molecular grade water, that is used as negative control in qPCR assay to check for the

absence of contaminant in the qPCR mix
3.9
cycle threshold

number of qPCR cycles required for the fluorescent signal to cross the threshold (i.e. exceeds

background level)

Note 1 to entry: The Ct value is inversely proportional to the abundance of the target sequence.

4 Principle

This International Standard describes qPCR assay using fluorescent DNA binding dye as reporter.

This qPCR assay has been validated by an international ring test conducted with the SYBR Green, a

commonly used fluorescent DNA binding dye which binds all double–stranded DNA and can be detected

by measuring the increase in fluorescence throughout the cycle.

The method aims to measure the abundance of selected microbial gene sequences from soil DNA

extract. The method comprises four tasks and eight steps as summarized in Figure 1. According to

Reference [1], the three critical steps to be validated for each qPCR assay are as shown in Figure 1.

2 © ISO 2016 – All rights reserved
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SIST EN ISO 17601:2018
ISO 17601:2016(E)
Validation
Validation

Figure 1 — Main tasks and critical steps to estimate the abundance of selected microbial gene

sequences by qPCR assay

This International Standard describes qPCR assay based on the use of fluorescent DNA binding dye

®1)

which has been validated by an international ring test using SYBR Green qPCR. In Annex A,

1) SYBR Green is a registered trademark of Molecular Probes. This information is given for the convenience of

users of this document and does not constitute an endorsement by ISO of the product named. Equivalent products

may be used if they can be shown to lead to the same results.
© ISO 2016 – All rights reserved 3
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SIST EN ISO 17601:2018
ISO 17601:2016(E)

information about TaqMan qPCR assay not tested in the international ring test are given. The

first task is made of three steps describing the design of optimal amplicon for qPCR (step one), the

preparation of qPCR standards (step two), and the procedure to calibrate the qPCR assay (step three).

The second task includes two additional steps describing the procedures to prepare soil DNA samples

(step four) and to test for the presence of qPCR inhibitors in soil DNA samples (step five). The third

task is constituted of a single step describing the protocol to perform qPCR assay (step six). Finally, the

fourth task is made of two steps, one describing the procedure to validate qPCR assays (step 7) to check

the quality of qPCR assay and another one describing the different options to calculate the number of

sequences of the gene of interest copy from cycle threshold (Ct) obtained from the analysis of qPCR

amplification plots (step 8).
5 Test materials
5.1 DNA

5.1.1 DNA, extracted from pure bacterial and fungal isolates using classical extraction procedures or

by using commercial kit to extract genomic DNA.
5.1.2 Soil DNA, extracted from aliquots of soil according to ISO 11063.
5.2 Bacteria
5.2.1 Escherichia coli strain, usually used for cloning of PCR product.
5.3 Plasmid

5.3.1 Cloning vector, usually used for cloning of PCR product in competent Escherichia coli.

5.4 Enzyme
5.4.1 Taq polymerase.
5.4.2 T4 DNA ligase.
5.4.3 T4 gene T32.
5.4.4 Bovine serum albumin (CAS No. 9048-46-8).
5.5 Chemicals
5.5.1 Ampicilline sodium, C H N NaO S (CAS No. 69-52-3).
16 18 3 4
5.5.2 Boric acid, BH O (CAS No. 10043-35-3).
3 3
5.5.3 Deoxynucleotide solution, dNTPs.
5.5.4 SYBR Safe DNA gel stain.

2) TaqMan is a trademark of Roche Molecular Systems, Inc. This information is given for the convenience of

users of this document and does not constitute an endorsement by ISO of the product named. Equivalent products

may be used if they can be shown to lead to the same results.
4 © ISO 2016 – All rights reserved
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SIST EN ISO 17601:2018
ISO 17601:2016(E)
5.5.5 Ethylenediaminetetraacetic acid disodium salt (EDTA), C H N O Na·2 H O
10 14 2 8 2 2
(CAS No. 6381-92 6).
5.5.6 Glucose, C H O (CAS No. 50-99-7).
6 12 6
5.5.7 Chlorhydric acid, HCl (CAS No. 7647-01-0).
5.5.8 IPTG, Isopropyl-Beta-D-Thiogalactopyranoside, (CAS No. 367-93-1).
5.5.9 Magnesium chloride, MgCl (CAS No. 7786-30-3).
5.5.10 Magnesium sulfate, MgSO (CAS No. 7487-88-9).
5.5.11 Molecular-biology-grade water, H O.
5.5.12 Potassium chloride, KCl (CAS No. 7447-40-7).
5.5.13 Sodium chloride, NaCl (CAS No. 7647-14-5).
5.5.14 Tris[hydroxymethyl]aminomethane, C H NO (CAS No. 77-86-1).
4 11 3

5.5.15 X-Gal, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside, (CAS No. 7240-90-6).

5.6 Product for bacterial culture medium
®3)
5.6.1 Bacto tryptone , enzymatic digest of casein.
5.6.2 Yeast extract powder (CAS No. 8013-01-2).
5.7 Buffer and reagents

5.7.1 Ampicilline solution, 2 g of ampicilline sodium in 4 ml of 0,22 µm filter sterilized H O. Adjust to

20 ml with sterilized H O, prepare 1 ml aliquots, and store at -20 °C.
-1 -1

5.7.2 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.

5.7.3 SYBR Safe™ DNA gel stain, dilute 10,000X SYBR Safe™ gel stain in TBE buffer × 1.

5.7.4 IPTG stock solution, 1 g of IPTG in 8 ml of H O. After careful mixing, the solution is adjusted to

10 ml and sterilized under security microbiology post. Prepare 1 ml aliquot of IPTG and store at -20 °C.

5.7.5 Solid LB medium, 10 g of Bacto tryptone , 5 g of yeast extract, 5 g of sodium chloride, and 15 g

of agar in 1 000 ml of H O. After autoclaving for 20 min at 120 °C, 1 ml of ampicilline stock solution at

100 mg·ml is added to LB medium and plated in Petri dishes (20 ml) under a security microbiology

post. 100 µl of IPTG solution are plated on solid LB-ampicilline medium. When IPTG solution is entered

in LB-ampicilline medium, 20 µl of X-Gal solution is plated on solid LB-ampicilline medium. Solid LB

medium is stored at 4 °C until its use.

3) Bacto tryptone is the trademark of a product supplied by Difco Laboratories. This information is given for

the convenience of users of this document and does not constitute an endorsement by ISO of the product named.

Equivalent products may be used if they can be shown to lead to the same results.

© ISO 2016 – All rights reserved 5
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SIST EN ISO 17601:2018
ISO 17601:2016(E)

5.7.6 SOC medium, 20 g of Bacto tryptone , 5 g of yeast extract, 0,58 g of NaCl, 0,95 g of MgCl , 2,46 g

of MgSO , and 3,60 g of glucose in 1 l H O. Sterilize by 20 min autoclaving at 120 °C. Prepare 950 ml

4 2
aliquots and store at -20 °C.
-1 -1

5.7.7 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.

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

1 000 ml of H O.
5.7.8 TBE buffer × 1, 100 ml of TBE buffer × 10 in 900 ml of H O.
-1 -1

5.7.9 TE buffer × 10, pH 8,0, 100 ml of 1 mol·l Tris-HCl pH 8,0, 20 ml of 50 mmol·l EDTA pH 8,0 in

880 ml of molecular grade water.
5.7.10 TE buffer × 1, 100 ml of TE buffer × 10 in 900 ml of H O.
5.7.11 X-gal solution, 250
...

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