OHS026
Safe Work Procedure
Faculty/Division
School/ Divisional Unit
Medicine
St. George Clinical School
Document number
STGCL.SWP.35.1
Initial Issue date
14/04/2010
Current version
1.0
Current Version
Issue date
14/04/2010
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: Preparation of buffers for use in SDS-PAGE and Western blotting
Description: This safe work procedure outlines the safe procedure for preparation of buffers used in
SDS-PAGE and Western Blotting
Associated risk assessment title and location: STGCL.RA.35.1
Describe the activity or process
This SWP outlines the procedure for preparing four buffers used in SDS-PAGE and Western blotting. All
relevant MSDSs and the attached Risk Assessment must be read prior to the task being undertaken.
Procedure:
Before making any of these buffers, wear Long sleeved laboratory Gown, Splash Goggles,
appropriate gloves (for Methanol & Hydrochloric Acid use Laminate gloves or Nitrile Gloves) and
closed footwear.
A.
1.
2.
3.
4.
Running Buffer (also called Laemmli Buffer B) (1X Stock)
Weigh out 188 g Glycine and 30.2 g Tris base into a 1 L beaker.
Add 600 mL of Milli Q water and stir gently until dissolved.
Add 100 mL of a 10% SDS solution (100 g/L)
Make up to 1 L with Milli Q water. This is 10X Running Buffer and it is normally stored as 10X and
diluted to 1X immediately prior to use.
5. Dilute the 10X Running Buffer by adding 100ml of 10X Running Buffer to 900ml of Milli Q water and mix
well.
6. Any other volume of 10X Running Buffer can be made as required as long as the same ratios of Running
Buffer to Milli Q water are used.
7. Running Buffer is also available commercially and can be purchased instead of making up the solution.
B.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Transfer Buffer (1X Stock)
Weigh out 144 g Glycine and 30 g Tris base into a 1 L beaker.
Add 600 mL of Milli Q water and stir gently until dissolved.
Make up to 1 L with Milli Q water (this is a 10X stock).
1X Stock is made up fresh as required.
For 1 Litre 1X Stock, take 100ml of 10X solution and 700ml of Milli Q water to the Fume Cupboard and
switch the fume cupboard on.
Use the Safety Bottle Carrier to transport Methanol from the Chemical Store to the fume cupboard.
Decant 200mL of methanol in the fumehood and add to the 100ml of 10X Transfer Buffer.
Add a further 700ml of Milli Q water, close the bottle and mix well.
Remove all items from the fume cupboard and switch it off (using safety carrier bottle for the Methanol).
Any other volume of 1X Transfer Buffer can be made as required as long as the same ratios of Transfer
Buffer to Milli Q water to Methanol are used.
Transfer Buffer is also available commercially and can be purchased instead of making up the solution.
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Safe Work Procedure
Date Effective: 01/01/2007
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Describe the activity or process
C. Phosphate Buffered Saline (PBS) (1X Stock)
Phosphate Buffered Saline can be made using PBS Tablets that are available commercially or
Phosphate Buffered Saline can be purchased ready made.
Using PBS Tablets
1. To make 1 Litre of 1X Phosphate Buffered Saline, place 10 PBS Tablets (Amresco PBS tablets,
Biotechnology Grade, Code: E404-200TABS) in a container and make up to 1 litre with Milli Q water.
2. Mix with magnetic stirrer / stir bar or alternatively allow it to dissolve without mixing
Phosphate Buffered Saline is also available commercially and can be purchased readymade rather than
making up the solution.
D. Phosphate Buffered Saline with 0.1% Tween 20 (PBST) (1X Stock)
1. Use Phosphate Buffered Saline (as per C above).
2. Tween 20 is viscous and is difficult to pipette a small volume of neat Tween 20 accurately, therefore
make 100 ml of a 1 in 10 dilution of Tween 20 first by adding 10 ml of Tween 20 to 90 ml water and
mixing well. Add 10ml of the 1 in 10 stock dilution of Tween 20 to 1 litre Phosphate Buffered Saline and
mix well.
E. Tris Buffer Saline Tween (50 mM Tris, 150 mM Sodium Chloride with 0.1% Tween 20)
1. Make 1 Molar Stock Solution of Tris-Hydrochloric Acid pH 7.4 and 5 Molar Stock Solution of Sodium
Chloride as follows:
2. 1 Molar Tris-Hydrochloric Acid Stock Solution: Weigh 121.14 g of Tris and add to a container. Add
approximately 700 – 800ml of Milli Q water and dissolve the Tris using a magnetic stirrer.
Using a
calibrated pH Meter, adjust the pH of the Tris Solution to pH 7.4 using 5M Hydrochloric Acid. Make up to
1 Litre with Milli Q Water.
3. 5 Molar Sodium Chloride Stock Solution: Weigh 292.2 g of Sodium Chloride and add to a container.
Add approximately 700 – 800ml Milli Q Water and dissolve using a magnetic stirrer. Make up to 1 Litre
with Milli Q Water.
4. Add 50ml of 1Molar Tris-Hydrochloric Acid Stock Solution to a container.
5. Add 30ml of 5 Molar Sodium Chloride Stock Solution and 10ml of the 1 in 10 stock dilution of Tween 20
(See D2) to the same container and mix well.
6. Make up to 1 Litre with Milli Q Water and mix.
List all resources required including plant, chemicals,
personal protective clothing and equipment, etc

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





Balance, Beakers, measuring cylinders, magnetic stirrer & stir bars, storage containers
Laboratory Gown, Splash Goggles, appropriate gloves
Glycine
Tris base (NB: this is a hazardous substance)
Sodium dodecyl sulphate (SDS) (NB: SDS is flammable and hazardous, and at 10% is an irritant)
Methanol (NB: this is a hazardous substance & Dangerous Good) - use Laminate or Nitrile Gloves
Tween 20 (NB: this is potentially hazardous)
Hydrochloric Acid is a Dangerous Good, is Corrosive, can cause burns and is an Irritant (use Laminate or
Nitrile Gloves).
 Sodium Chloride, Phosphate Buffered Saline Tablets (This is an irritant to the eyes, skin and mucous
membranes),
List potential hazards and risk controls including
specific precautions required
Hazard: Chemicals in particular Tris, Sodium Dodecyl Sulphate, Methanol and Hydrochloric Acid
Risk Control 1: Wear Long sleeved Laboratory Gown, appropriate laminate or Nitrile gloves, Closed
footwear, Splash Goggles
Risk Control 2: Use Safety Bottle carrier to carry Methanol from the Chemical Store to the Fumehood and
back
Risk Control 3: Use of fume cupboard for decanting Methanol
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Date Effective: 01/01/2007
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Current Version: 1.2, 15/08/2007
List emergency shutdown instructions
N/A
List clean up and waste disposal requirements
If a spillage occurs, follow the Spillage Procedure
All buffers containing hazardous substances / dangerous goods have to be collected in designated
chemical waste cubes and are disposed of as chemical waste through the UNSW Chemical waste disposal
system.
List legislation, standards and codes of practice
used in the development of the SWP
MSDSs for SDS, Tris base, methanol, Tween 20, Hydrochloric Acid, PBS tablets
Supervisory approval, training, and review
Supervisor: Prof S. Krilis (Immunology)
Signature:
Supervisor: Prof B. Chong (Medicine)
Signature:
Supervisor:
Signature:
Supervisor:
Signature:
Supervisor:
Signature:
Plant custodian: Prof S. Krilis
Signature
List competency required – qualifications, certificates, licencing, training - eg course or instruction:
Training in this Safe Work Procedure, Hazardous Substance Training
SWP review date: April 2012
Responsibility for SWP review: Qi Miao
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Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007