OHS026
Safe Work Procedure
Faculty/Division
Medicine
Document number
POWCS.ORC.SWP M33
School/ Divisional Unit
POWCS/ORC
Initial Issue date:
02/06/2008
Current version
2
Current Version
Issue date: 29/01/2010
Next review date
01/12/2011
The Writing Safe Work Procedures Guideline (OHS027) should be consulted to assist in the completion of this form.
Safe Work Procedure Title and basic description
Title:
DNA ligation
Description:
This SWP describes the correct procedures to be followed when carrying out the ligation of DNA
fragments from different sources to form a new recombinant DNA molecule.
Associated risk assessment title and location: :
: POWCS.ORC.RA M33
Describe the activity or process
This SWP describes the correct procedures to be followed when carrying out the ligation of DNA
fragments from different sources to form a new recombinant DNA molecule.
This methodology involves the use of hazardous substances. The attached Risk Assessment and all
relevant MSDSs must be accessed and read before the procedure is carried out.
This document cross-references other SWPs. Be aware that the hazards associated with the
procedures described in those documents will not be detailed here. It is the individual’s responsibility
to read the appropriate SWPs and their associated Risk Assessments before beginning this work.
Background:
The term recombinant DNA encapsulates the concept of recombining fragments of DNA from different
sources into a new DNA molecule. Joining linear DNA fragments together with covalent bonds is called
ligation. More specifically, DNA ligation involves creating a phosphodiester bond between the 3’
hydroxyl of one nucleotide and the 5’ phosphate of another.
The enzyme used to ligate DNA fragments is T4 DNA ligase, which originates from the T4
bacteriophage. This enzyme will ligate DNA fragments having overhanging, cohesive ends that are
annealed together, as in the EcoRI example below – this is equivalent to repairing “nicks” in duplex
DNA. T4 DNA ligase will also ligate fragments with blunt ends, although higher concentrations of the
enzyme are usually recommended for this purpose.
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Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
Describe the activity or process
Procedure:
1. Ligation reactions of plasmid vectors and DNA inserts are routinely carried out in a final volume of
10 – 20 µL. Generally, fragments to be inserted are in 2 – 4-fold molar excess over the vector
(three ratios of insert to vector are are employed to ensure optimal results: 1:1, !:3 and 3:1).
2. The vector and the insert are prepared by restriction enzyme digestion (see SWP M31) and gel
purification (see SWP M32).
3. Decide upon the final volume of the ligation reaction (10 µL or 20 µL), and calculate the volumes
of each component. Add water, then 10x buffer, then 5 U T4 DNA ligase, then the vector and the
insert to a sterile 1.5 mL tube. Also prepare a matched control sample by omitting the insert. In
the example below, the Survivin promoter gene prepared by BgIII/HindIII restriction enzyme
digestion is being inserted into the pGL3 plasmid, linearised by BgIII/HindIII restriction enzyme
digestion:
4.
5.
6.
7.
8.
Control
Ligation
pGL3 (150 ng/µL)
1 µL
1 µL
Survivin (40 ng/µL)
---
7 µL
10x buffer
1 µL
1 µL
Ligase (5 U/µL)
1 µL
1 µL
Water
7 µL
---
Total
10 µL
10 µL
Incubate overnight in an ice/water bath (i.e. a slurry) in an esky; the esky can be left at room
temperature. The optimal incubation temperature for T4 DNA ligase is 16oC and when very high
efficiency ligation is desired (e.g. making libraries) this temperature is recommended. However,
the ligase is active at a broad range of temperatures, and for routine purposes such as subcloning, convenience often dictates incubation time and temperature. Ligations performed at 4 oC
overnight or at room temperature for 30 – 120 min usually work well.
Incubate ligations at room temperature for 30 min
Store at -20oC, or carry out transformations in DH10B cells (see SWP M10)
Pick single bacterial colonies, and carry out miniprep DNA preparations (see SWP M6)
Check the ligation by carrying out restriction enzyme digestions (see SWP M33)
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Safe Work Procedure
Date Effective: 01/01/2007
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Current Version: 1.2, 15/08/2007
List all resources required including plant, chemicals,
personal protective clothing and equipment, etc


Restriction enzyme digested and gel purified DNA inserts and vectors (see SWP GT6 and SWP GT7)
T4 DNA ligase (Fermentas, cat. # EL0014, 5 U/µL), supplied with 10x buffer (NB: - this is a hazardous
substance)
 Molecular biology grade water
List potential hazards and risk controls including specific
precautions required
T4 DNA ligase is a mixture which contains hazardous ingredients with concentration less than 1% (HMIS
rating 1*). It may cause skin irritation if it comes in contact with skin. It may cause eye irritation if there is
eye contact. Wash eye with copious amount of water in case of contact. Read MSDS for T4 DNA ligase
before handling solution.
List emergency shutdown instructions
Stop work, if possible, secure reagents and the facility appropriately, warn others, remove PPE and leave
the premises to go to a safe location.
List clean up and waste disposal requirements
Throw all eppendorf tubes in GMO waste container. Discard tube containing ligase buffer and T4 DNA
ligase eppendorf tubes in cytotoxic waste container.
SWP P4 and P5 for GMO-waste management. Spill SWP A4 for spill mangement
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Safe Work Procedure
Date Effective: 01/01/2007
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Current Version: 1.2, 15/08/2007
List legislation, standards and codes of practice used in the
development of the SWP
MSDS for 10x ligation buffer for T4 DNA ligase (cat. # EL0014)
 Guidelines for dealings with Genetically modified organisms in accordance with the gene
technology legislation
 Section 4 Classification of laboratory practices and procedures (AS/NZS 2243.3:2002)
 Management of biological spills: Guidelines Version: 2 (section 5, AS/NZS 2243.3:2002)
 Appendix E: Chemical disinfectants (AS/NZS 2243.3:2002)
 Laboratory Cleaning. Section 8 (AS/NZS 2243.3:2002)
 Waste Disposal Section 9 (AS/NZS 2243.3:2002)
 Gene technology Procedure version 0.1, 01/11/2006 www.ogtr.gov.au/pubform/index/htm
 www.riskman.unsw.edu.au/ohs/genetech or
(http://www.hr.unsw.edu.au/ohswc/ohswc_home.html)
AS/NZ 2243.3:2002 Safety in Laboratories
Section 4: classification of laboratories, practices and procedures.
AS/NZ 2243.3:2002 Safety in Laboratories
Section 6: general precautions and special equipment.
Management of Biological Hazardous Spills or Accidental Release of Biological Agents
(UNSW Draft v2, March 2001).
AS/NZ 2243.3:2002 Safety in Laboratories
Section 5: laboratory spills.
AS/NZ 2243.3:2002 Safety in Laboratories
Appendix E: chemical disinfectants.
AS/NZ 2243.3:2002 Safety in Laboratories
Section 8: laboratory cleaning.

AS/NZ 2243.3:2002 Safety in Laboratories
Section 9: waste disposal
Supervisory approval, training, and review
Supervisor: Dr Aparajita khatri
Signature:
Plant custodian:
Signature
List competency required – qualifications, certificates, licencing, training - eg course or instruction:
 UNSW PC-2 training
 ORC PC-2 training
 Demonstration of techniques by competent individual
SWP review date:
Responsibility for SWP review:
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Safe Work Procedure
Date Effective: 01/01/2007
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Current Version: 1.2, 15/08/2007