OHS026
Safe Work Procedure
Faculty/Division
Medicine
Document number
SOMS.CGM.SWP038
School/ Divisional Unit
School of Medical Sciences
Initial Issue date
30/6/09
Current version
1.0
Current Version
Issue date 30/6/09
Next review date
30/6/12
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: 12% High resolution SDS gel electrophoresis for Myosin Heavy Chains
Description: Separating Myosin Heavy Chains isoforms by denaturing SDS polyacrylamide gel
electrophoresis.
Associated risk assessment title and location: 38_RA_MyHC gel electrophoresis_CL
Describe the activity or process
NOTE: Read all MSDS’s before starting experiment
1. Set up plates-rinse with dH20, 100% ethanol
2. Leave plates to dry
Make up fresh Acrylamide-Bis solutions
 30% Acrylamide (200:1) solution
(i.e. 5.97g Acrylamide and 0.03g Bis make up to 20 mls)
 30% Acrylamide (37.5:1) solution
3. Make up Ammonium Persulphate solution (APS) in eppendorf: (0.1g APS +1 ml dH20)
4. Make up resolving gel (follow table below), leave to set 1 hour
5. Make up lower buffer: 30.29g Tris + 142.6g Glycine and fill to 5L with stirrer
6. Make up upper buffer (and put in fridge): separate 1L from lower buffer, add below and put in
fridge then add 20 ml 10% SDS to lower buffer, 10 ml 10% SDS to upper buffer + 0.3g DTT
7. Place cooler in tank (set to 5-6 degrees): should measure 10-12 degrees
Make up Stacking Gel (follow table below) leave to set for 2 hours
12 % SDS-PAGE for separation of Myosin Heavy Chain Gels
12% Resolving gel
4% Stacking gel
30ml (2 gels)
20 ml (2gels)
100% glycerol
9 ml
6 ml
30% Acrylamide- Bis (200:1)
12 ml
_
30% Acrylamide-Bis (37.5:1)
_
2.6 ml
1.5 M Tris (pH8.8)
4 ml
_
0.5 M Tris (pH6.7)
2.8 ml
1M Glycine
3 ml
_
10% SDS
1.2 ml
0.8 ml
100mM EDTA (pH 7.0)
0.8 ml
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Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
Distilled H2O
0.5
6.7
10% APS
0.3 ml
0.2 ml
TEMED
4.5 ul
4.5 ul
8. For Normal run set limit voltage to 150V and limit current to 20 mA, for pulse (20s on/off) set
limit current to 20 mA.
9. Stain gel in Coomassie Brillant Blue solution
List all resources required including plant,
chemicals, personal protective clothing and
equipment, etc
Equipment:
Gel Pouring Apparatus
Glass Plates and Spacer Bars
Plastic Lane Combs
Plastic Falcon Tubes
Fume Hood
Chemicals:
30% Acrylamide (200:1) solution
30% Acrylamide (37.5:1) solution
10% Ammonium persulphate (APS)
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)
Tris and Glycine for Upper and Lower Buffers
Coomassie Brilliant Blue Solution
Personal Protective Equipment:
Safety Goggles
Lab Coat/Gown
Face Mask
List potential hazards and risk controls including
specific precautions required
Acrylamide – Toxic, carcinogen/mutagen, neurotoxin - may cause cancer and heritable genetic damage. Toxic in
contact with skin and if swallowed. Danger of serious damage to health by prolonged exposure through inhalation, in
contact with skin or if swallowed. May cause eye irritation and allergic skin reaction. Polymerization may occur from
excessive heat or contamination. Wear safety goggles where splashing is possible. Wear enclosed shoes, nitrile
gloves, and gown.
Bis-acrylamide – Irritant. Irritating to eyes, respiratory system and skin. Harmful if swallowed, inhaled or absorbed.
Toxic if ingested. Wear safety goggles where splashing is possible. Wear enclosed shoes, gloves and gown.
TEMED - Corrosive. May flame on contact with heat or fire. Moderately toxic via oral route. Continuous exposure of
low levels of vapour can cause a blurring of vision called "Blue Haze". Wear gloves, mask, goggles, and gown.
Keep away from ignition sources.
Ammonium Persulfate (APS) – Will cause irritation to eyes, which results in corneal injury, and to respiratory
system. May cause irritation to skin. Wear safety goggles, nitrile rubber gloves, gown and enclosed shoes.
SDS- Respiratory, skin and eye irritant. Harmful if swallowed, inhaled or absorbed through the skin. Severe eye
irritant. Nuisance dust, but may also cause sensitization if inhaled. While working with SDS in solution, wear
gloves, gown, and mask where risk of splashing is possible. SDS is kept in small (<100ml) volumes in the lab.
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Safe Work Procedure
Date Effective: 01/01/2007
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Current Version: 1.2, 15/08/2007
Tris- Low toxicity- Skin, eye, and respiratory irritant. Decant in well ventilated areas. Wear gown and gloves when
handling.
Glycine- Low toxicity- eye irritant. Decant in well ventilated areas. Wear gown and gloves when handling.
Glycerol- Low toxicity. Glycerol may present a hazard with direct eye contact or prolonged skin contact. Wear
gloves and gown.
Coomassie Brilliant Blue- Low toxicity. Wear gloves and gown, and goggles if measuring volumes greater than 1g.
Power pack usage- All electrical items are hazardous. Because of the nature of this electrophoresis, there is a risk of
liquid (ie buffers) contacting the power pack.
List emergency shutdown instructions
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 APS and TEMED and
disposed in the cytotoxic waste container.
List legislation, standards and codes of practice
used in the development of the SWP
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NSW OHS Act 2000
NSW OHS Regulation 2001
Safe Work Procedure Form (OHS026)
Australia Dangerous Goods Code.
AS/NZS 2243.2:2006. Safety in laboratories. Part 2: Chemical aspects
Australian Standard AS2243.3-2002. Safety in laboratories. Part 3: Microbiological aspects and containment
facilities.
AS/NZS 2161.1:2000 Occupational Protective Gloves – Selection, Use and Maintenance
AS/NZS 1336:1997 Recommended Practices for Occupational Eye Protection
Supervisory approval, training, and review
Supervisor: Edna Hardeman
Signature:
Plant custodian:
Signature
List competency required – qualifications, certificates, licencing, training - eg course or instruction:
Training as per Training Needs Analysis, Induction to the Lab, Training in this SWP
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Page 3 of 3
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007