OHS026
Safe Work Procedure
Faculty/Division
Medicine
Document number
SOMS.CGM.SWP041
School/ Divisional Unit
School of Medical Sciences
Initial Issue date
23rd June 2009
Current version
1.0
Current Version
Issue date
23rd June 2009
Next review date
23rd June 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: Sodium Dodecyl Sulphate - Polyacrylamide gel for electrophoresis (SDS-PAGE)
Description: Separating cell extracts in a denaturing polyacrylamide gel by electropheresis.
Associated risk assessment title and location: 41_RA_Pouring Acrylamide Gels_SW
Describe the activity or process
NOTE: Read all MSDS’s before starting experiment
Polyacrylamide – carcinogenic and neurotoxic, wear protective clothing and gloves, once
set, can be disposed of in the purple cytotoxic bin
Solution Required
 30% Acrylamide/Low-Bis solution (Refer to SWP “Making 30% Low-Bis Acrylamide
Solution)
Polyacrylamide – carcinogenic and neurotoxic, wear protective clothing and gloves, once
set, can be disposed of in the purple cytotoxic bin
 10% Ammonium persulphate (AMPS)
 N,N,N',N'-Tetramethylethylenediamine (TEMED)
Harmful if inhaled or swallowed. Corrosive - causes burns. Irritant. Wear protective
clothing, face mask and gloves. Addition of TEMED to acrylamide gel solution should be
carried out in a well ventilated area or a fume hood.
 Resolving buffer (1.5M Tris.Cl pH 8.8)
 Stacking buffer (0.5M Tris.Cl pH 6.8)
 10% (w/v) Sodium dodecyl sulfate (SDS)
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Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
Description:
12.5 % Low-Bis Acrylamide Resolving Gel:
1 gel
2 gels
4 gels
5ml
10ml
30% Acrylamide- 2.5 ml
Low Bis
1M Tris pH8.8
2.25ml
4.5ml
9ml
Water
1.14ml
2.3ml
4.6ml
10% SDS
TEMED
10% APS
Prepare PAGE apparatus following manufacturer’s instructions.
 Combine H2O, resolving buffer, acrylamide monomer.
 Allow to warm to room temperature (degassing optional).
 Add SDS, TEMED, and AMPS and mix well.
Pour gel to within 2cm (4.5cm for large Bio-Rad apparatus or 2.5cm for smaller gels) of the top
of plates, overlay with water or ethanol and allow to polymerise for approximately 30 mins.
Can now leave gel overnight if covered with Parafilm.
Remove overlay, rinse gel with distilled water, dry with filter paper, and pour stacking gel.
12.5 % Low-Bis Acrylamide Stacking Gel:
1 gel
30%
2 gels
Acrylamide
4 gels
1.33ml
Bis
1M Tris pH6.8
Water
1.25ml
1.45ml
2.9ml
7.25ml
10% SDS
TEMED
10% APS




Combine H20, stacking buffer, and acrylamide monomer
Allow to warm to RT (degassing optional).
Add SDS, TEMED, AMPS and mix well.
pour stacking gel, insert comb and allow to polymerise for approximately 30 minutes.
Gels can be stored at 4oC by wrapping in paper towel soaked in transfer buffer and covered in
plastic wrap.
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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
Equipment:
Gel Pouring Apparatus
Short and Spacer Plates
Plastic Lane Combs
Plastic Falcon Tubes
Fume Hood
Chemicals:
30% Acrylamide/Low-Bis solution
10% Ammonium persulphate (AMPS)
N,N,N',N'-Tetramethylethylenediamine (TEMED)
Resolving buffer (1.5M Tris.Cl pH 8.8)
Stacking buffer (0.5M Tris.Cl pH 6.8)
10% (w/v) Sodium dodecyl sulfate (SDS)
Personal Protective Equipment:
Safety Goggles
Lab Coat/Gown
Face Mask (for addition of TEMED to acrylamide solution)
List potential hazards and risk controls including
specific precautions required
Potential Hazards:


Contact and absorption of acrylamide via contact of solution with skin or splashing into
eyes.
Inhalation of TEMED via vapors
Risk Controls and Precautions:


Gloves, Safety goggles and lab coat should be worn when handling acrylamide solutions
and TEMED
Making of polyacrylamide gels should be carried out in a well ventilated area, preferably
in a fume hood.
List emergency shutdown instructions
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Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
List clean up and waste disposal requirements
Spillage of any solution used for the making of polyacrylamide gels should be cleaned up using
the spill kit located in Rm 502. Paper towel used to mop up excess acrylamide solution should
be disposed in the cytotoxic waste container
Excess acrylamide solution should be polymerized with addition of AMPS and TEMED and
disposed in the cytotoxic waste container.
List legislation, standards and codes of practice used
in the development of the SWP
NSW OHS Act 2000
NSW OHS Regulation 2001
AS/NZS 2243.2:2006. Safety in laboratories. Part 2: Chemical aspects
AS/NZS 2161.1:2000 Occupational Protective Gloves – Selection, Use and Maintenance
AS/NZS 1336:1997 Recommended Practices for Occupational Eye Protection
Sodium Dodecyl Sulphate - Polyacrylamide gel electrophoresis (SDS-PAGE)
Children’s Cancer Institute Australia Standard Operating Procedure (No. CCIA-LB-57)
Supervisory approval, training, and review
Supervisor: Peter Gunning
Signature:
Plant custodian:
Signature
List competency required – qualifications, certificates, licencing, training - eg course or instruction:
Training as per Training Needs Analysis, Induction into the Lab, Training on this SWP.
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Page 4 of 4
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007