OHS026
Safe Work Procedure
Faculty/Division
School/ Divisional Unit
Medicine
ST. George Clinical School
Document number
STGCL.SWP.51.1
Initial Issue date:
22/04/10
Current version
1.0
Current Version
Issue date: 22/04/10
Next review date
April 2012
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: STGCL.RA.51.1
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.
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 refers to 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 and also fragments with blunt ends.
Example:
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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 µL. Generally, fragments to be inserted are in 2 – 4-fold molar excess over the vector (Other
ratios of insert to vector can be employed to ensure optimal results: eg: 1:1, 1:3 and 3:1).
2. The vector and the insert are prepared by restriction enzyme digestion (see SWP ) and gel
purification (see SWP ).
3. Calculate the volumes of each component. Add water, then 10x buffer, then 6 U of 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. Example of inserting fragment X prepared by BgIII/HindIII restriction enzyme
digestion into the pGL3 plasmid, linearised by BgIII/HindIII restriction enzyme digestion:
4.
5.
6.
7.
Control
Ligation
pGL3 (150 ng/µL)
1 µL
1 µL
Fragment X (40 ng/µL)
---
6 µL
10x Ligation buffer
1 µL
1 µL
Ligase (3 U/µL)
2 µL
2 µL
Water
6 µL
---
Total
10 µL
10 µL
Incubate 4 hours or overnight in an ice/water bath (i.e. a slurry) in an esky; the esky can be left at
room temperature. NB: The optimal incubation temperature for T4 DNA ligase is 16oC and when
very high efficiency ligation is desired this temperature is recommended. However, the ligase is
active at a broad range of temperatures. For routine subcloning, ligations performed at 4 oC
overnight or at room temperature for 30 – 120 min usually work well.
Store at -20oC for later use, or carry out transformations in appropriate competent cells (see
STGCL.SWP.49.1)
Pick single bacterial colonies, and carry out miniprep DNA preparations
Check the ligation by carrying out restriction enzyme digestions (see STGCL.SWP.45.1)
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Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
List all resources required including plant, chemicals,
personal protective clothing and equipment, etc
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Restriction enzyme digested and gel purified DNA inserts and vectors
T4 DNA ligase (Promega, cat. # M1804, 3 U/µL), supplied with 10x buffer
Milli Q sterile water
Esky or fridge
Competent cells
37 C incubator
SOC medium
LB plates containing appropriate antibiotic
37 C shaker
42 C water bath
List potential hazards and risk controls including specific
precautions required
There are no hazardous substances used in the ligation reaction. Standard laboratory practices including
the use of PPE must be followed. Risks associated with linked procedures such as transformation (see
STGCL.SWP.49.1), DNA mini-preparation and restriction enzyme digests (see STGCL.SWP.45.1) are
listed in the corresponding SWP/RA
List emergency shutdown instructions
N/A
List clean up and waste disposal requirements
If a spillage occurs, follow the Spillage Procedure
Disposed of all Eppendorf tubes as laboratory waste in the bins lined with yellow bags provided
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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
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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.
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AS/NZ 2243.3:2002 Safety in Laboratories
Section 9: waste disposal
Supervisory approval, training, and review
Supervisor: Prof B. Chong
Signature:
Supervisor:
Signature:
Supervisor:
Signature:
Supervisor:
Signature:
Plant custodian: Prof B. Chong
Signature
List competency required – qualifications, certificates, licencing, training - eg course or instruction:
 Training in this Safe Work Procedure & Corresponding Risk Assessment
 Demonstration of techniques by competent individual
SWP review date: April 2012
Responsibility for SWP review: Jose Perdomo
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Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007