OHS026
Safe Work Procedure
Faculty/Division
School/ Divisional Unit
Medicine
STGCL/MED
Document number
STGCL.SWP.69.1
Initial Issue date
24/04/2010
Current version
1.0
Current Version
Issue date
24/04/2010
Next review date
24/04/2012
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: Preparation of polyacrylamide gels
Description: Polyacrylamide gels are used in electrophoresis mobility shifting assay and protein separation experiments.
Associated risk assessment title and location: STGCL.RA.69.1 Risk assessment for preparation of polyacrylamide gel.
Describe the activity or process
This SWP describes the proper process for preparation of polyacrylamide gels which is used in the
electrophoresis mobility shifting assay and western blot experiment.
Procedure
A). Preparation of polyacrylamide gel for electrophoresis mobility shifting assay
1. Prepare 10x TBE buffer
 108g Tris base
 55g Boric acid
 7.4g EDTA (or 9.3g of Na2EDTA.2H2O), Ph to 8.2 in 900ml and make up to 1L with dH2O.
Store at room temperature.
2.
Prepare Gel mixture
Mix the following substances and buffers together in a 50ml tube and pour the mixture into the glass
plate sandwich (Proper procedures are shown below):
 8.5ml of 30% acrylamide (29:1) (Note: this is a hazardous substance. Use only in a
chemical fume hood)
 2.5ml 10x TBE buffer
 0.5ml 10% APS
 8.7ml water
 50ul TEMED
Gel apparatus
a. A gel sandwich is assembled with the singlescrew clamps
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b. Sandwiches are aligned using the casting stand’s
alignment slot and an alignment card
c. After gels are cast sandwiches are snapped onto the
gel running holder.
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B). Preparation of polyacrylamide gel for western blotting
1. Assemble the glass plate sandwich according to the following protocol:
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Describe the activity or process
2. Preparation of separating gel and stacking gel:
 Preparation of Separating gel buffer and Stacking gel buffer:
Separating buffer
Stacking buffer
Tris base
45.4g
15.15g
SDS
1.0g
1.0g
dH2O
Make up to 500ml
Make up to 500ml
pH adjustment
8.8
6.8
 Separating gel (make this mixture first. Add components in this order)
Mix the substances and buffers in the following table (shown for 8, 10 and 12% gels)
12%
Separating Buffer
30% Acrylamide
10% APS
TEMED
dH2O

8.1ml
6.75ml
180ul
18ul
1.65ml
10%
3.75ml
4.95ml
165ul
9ul
6.3ml
8%
7.5ml
4.05ml
165ul
9ul
7.2ml
Stacking gel (make this mixture after the separating gel has set. Add components in this
order)
3%
Staking Buffer
30% Acrylamide
10% APS
TEMED
dH2O
1.875ml
750ul
150ul
15ul
4.875ml
4%
4.5ml
1.62ml
90ul
13.5ul
3.45ml
5%
1.875ml
1.125ml
150ul
5.5ul
4.5ml
3. Transfer the separating gel mixture to the assembled sandwich chamber till 3/4 of the glass plate.
Overlay this with distilled water (this helps the gel to set evenly).
4. After the separating gel has set, remove water by tipping the whole assembly over the sink, and
removing the last traces of water with a tissue.
5.
Fill the space of the chamber with Stacking gel mixture and then insert the comb.
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List all resources required including plant,
chemicals, personal protective clothing and
equipment, etc
 Gloves
 Lab coat
 Safety glasses
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PROTEAN II xi | XL Vertical Electrophoresis Cells (Bio-Rad)
Whatman paper
30% acrylamide/bis 29:1 (BIO-RAD, 161-0156)
N,N,N’,N’,-Tetramethylethylenediamine (TEMED) ( Sigma, T9281)
10% Ammonium persulphate (APS; BIO-RAD, cat. # 161-0700)
SDS (BIO-RAD, cat. # 161-0302)
Tris (Amresco, 0497-1kg)
List potential hazards and risk controls including
specific precautions required
Hazards: Cuts from using glass plates
Risk Control: Training on this Safe Work Procedure and Practical Demonstration
Hazard: Handling Hazardous Substances / Dangerous Goods eg 30 % Acrylamide, Ammonium
Persulphate, SDS, Tris
Risk Control: Wear Lab Gown, Gloves and Lab Glasses and use Liquid Acrylamide in preference to
powder. Pour acrylamide in the fume hood
Hazard: Handling TEMED
Risk Control: Use Fumehood & wear Lab Gown, Gloves and Lab Glasses
List emergency shutdown instructions
Acrylamide:
In case of contact, immediately flush skin with plenty of water for at least 15 minutes while removing
contaminated clothing and shoes. Cover the irritated skin with an emollient. Cold water may be
used.Wash clothing before reuse.Thoroughly clean shoes before reuse. Get medical attention
immediately.
List clean up and waste disposal requirements
Polyacrylamide gel material generated during the process has to be disposed of as chemical waste.
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List legislation, standards and codes of practice
used in the development of the SWP
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MSDS for all the chemical used in this 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
Supervisory approval, training, and review
Supervisor: Prof. Beng Chong
Signature:
Supervisor:
Signature:
Supervisor:
Signature:
Supervisor:
Signature:
Supervisor:
Signature:
Supervisor:
Signature:
Plant custodian: Prof Beng Chong
Signature
List competency required – qualifications, certificates, licencing, training - eg course or instruction:
UNSW Laboratory Safety Awareness course
Practical demonstration
Training on this SWP
SWP review date:24/04/2012
Responsibility for SWP review: Shengyi LIU
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Date Effective: 01/01/2007
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Current Version: 1.2 , 15/08/2007