OHS017
OHS Risk Assessment and
Control Form
Risk assessment completed by:Dr S. Palmer
Staff/student number: 3283213
School/Unit: School of Medical Science, Neuromuscular and Regenerative
Faculty/Division: Medicine
Document number
SOMS.CGM.RA012
Medicine Research Unit
Initial Issue date
30/06/09
Current version
1.0
Current Version
Issue date 30/06/09
Next review date
30/06/12
For additional information refer to the OHS Risk Assessment and Control Procedure, the OHS Risk Rating Procedure and the Hierarchy of Risk Controls.
Risk Assessment title:: Electrophoretic Mobility Shift Assay (EMSA)
Step 1: Identify the activity
SOLUTIONS
0.1x TE
1M HEPES pH 7.9
3M KCl
0.5M EDTA
1M DTT
0.2M PMSF
Dialysis buffer (Brennan and Olson, 1990, Genes Dev 4:582-595)
Reagent
Quantity
Final Concentration
1M HEPES pH 7.9
15μl
15mM
3M KCl
33.3μl
100mM
100% glycerol
200μl
20%
0.5M EDTA
2μl
1mM
1M DTT*
0.5μl
0.5mM
0.2M PMSF*
2.5μl
0.5mM
H2O
746.7μl
*Add just prior to use
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Date Effective: 01/01/2007
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Current Version: 2.6, 16/07/2008
200mM benzamidine
1mg/ml pepstatin/leupeptin/aprotinin
10x Binding buffer
Reagent
1M HEPES pH 7.9
1M MgCl2
0.5M EDTA
0.1M DTT*
200mM Benzamidine*
1mg/ml Pepstatin*
1mg/ml Leupeptin*
1mg/ml Aprotinin*
0.2M PMSF*
H2O
*Add just prior to use
Quantity
12μl
4μl
1.6μl
4μl
8μl
4μl
4μl
4μl
2μl
36.4μl
Final Concentration
150mM
50mM
10mM
5mM
20mM
50μg/ml
50μg/ml
50μg/ml
5mM
Quantity
20μl
133.3μl
312
1.5μl
0.4μl
0.5μl
10μl
5μl
5μl
5μl
2.5μl
504.8μl
Final Concentration
20mM
400mM
25%
1.5mM
0.2mM
0.5mM
2mM
5μg/ml
5μg/ml
5μg/ml
0.5mM
5x TBE
0.5x TBE
Extraction buffer
Reagent
1M HEPES pH 7.9
3M KCl
80% glycerol
1M MgCl2
0.5M EDTA
1M DTT*
200mM Benzamidine*
1mg/ml Pepstatin*
1mg/ml Leupeptin*
1mg/ml Aprotinin*
0.2M PMSF*
H2O
*Add just prior to use
100mM KCl
1% NP40
10μl NP40 + 990μl dH2O. Store at 4C.
PROCEDURE
Annealing Oligonucleotides
Add oligos together in TE
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Heat oligo mix for 3 min @ 95-100oC in a heating block, then turn off heating block and allow to cool slowly to room temperature O/N
Radioactively Labeling Annealed Oligonucleotide
Mix reagents from Ambion DecaprimeII kit with annealed oligonucleatides
Incubate 10-15 mins at 37C on a hot-block.
Add 40μl TE buffer pH 8.
Centrifuge G-25 column placed into 1.5ml microcentrifuge tube at 3000 rpm for 1min.
Transfer column into a fresh 1.5ml microcentrifuge tube and load sample on top of dried beads of column.
Centrifuge at 3000 rpm for 2 mins.
Make up final volume of probe to 100μl with TE buffer, pH 8 (final concentration ~0.2ng/μl).
Estimate efficiency of labeling reaction by holding the GM tube at a reasonable distance from the tube containing the purified probe. Compare by experience with previous reactions.
Keep on ice if using that day, or store for longer periods at -20C.
Making Acrlyamide Gel
Wash the glass plates of the acrylamide gel electrophoresis system thoroughly with detergent, copious water and finally with 100% ethanol. Dry with Kimwipes.
Assemble the glass plates with spacers and mount in a position to allow gel pouring. Prepare a comb with the appropriate number of slots.
Gel mix
Reagent
Quantity
Final Concentration
30% Acrlylamide/bis (29:1)
5.33ml
4%
80% Glycerol
1.25ml
2.5%
5X TBE
4ml
0.5x
10% APS*
400μl
0.1%
TEMED*
40μl
H2O
29ml
*Add just prior to use
Mix the reagents in a beaker and add between the glass plates using a 10 or 20ml syringe.
Allow the gel to set for at least 30mins
EMSA Sample Preparation
The following reagents need to have these final concentrations per reaction:
Reagent
Quantity
KCl
40mM
Glycerol
5.25%
MgCl2
5mM
Nonidet P40 (NP40)
0.1%
Poly dI-dC
0.5μg
labeled oligo
~0.2ng
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Date Effective: 01/01/2007
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Current Version: 2.6, 16/07/2008
Example: In vitro translated protein (50ul reaction)
Reagent
Quantity
Dialysis buffer
10μl
10X binding buffer
4μl
100mM KCl
6μl
0.5μg/μl Poly dI-dC
1μl
1% NP40
4μl
Extract
4μl
Probe*
1μl
H2O
10μl
* Mix all reagents, then add probe and mix again.
Final Concentration
25mM KCl, 5% glycerol
5mM MgCl2
15mM KCl
0.5μg Poly dI-dC
0.1% NP40
~0.2ng labeled oligo
Running Samples on EMSA Gel
Incubate protein/DNA mixtures at room temperature for 20 min.
While incubating samples, pre-run gel in 0.5x TBE at 4C, 180V. The gel and buffer needs to be pre-cooled to this temperature well before running. This can be done with a buffer chiller or by
running in the cold room.
Load samples, run gel at 180V, 4C for approximately 3hrs (must be judged empirically)
Gel drying and X-ray film exposure
Dry gel onto a piece of Whattman paper (use gel drier at 80C for 1h). Be careful not to contaminate the dryer.
Expose gel to x-ray film (Hypercassette, MS intensifying screen & Kodak MS film for extra sensitivity), usually overnight, at -80C.
Describe the location:
Laboratory Rm 501, Fume hood Rm 525 and radiation suite Rm 403 in Wallace Wurth.
Step 2: Identify who may be at risk by the activity
A number of people may be at risk from any activity. This may affect the risk controls needed. These people may include fellow workers, visitors, contractors and the public. The location of the activity may affect the
number of people at risk.
Operator and those in the vicinity – particularly radiation hazard.
Steps 3 to 7: Identify the hazards, risks, and rate the risks
1. An activity may be divided into tasks. For each task identify the hazards and associated risks.
2. List existing risk controls and determine a risk rating using the UNSW Risk Rating Procedure.
3. Additional risk controls may be required to achieve an acceptable level of risk. Re-rate the risk if additional risk controls used.
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Risk Assessment and Control Form
Date Effective: 01/01/2007
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Current Version: 2.6, 16/07/2008
Tasks
Hazards
Associated risks
(Step 3)
(Step 4)
Risk rating with
existing controls *
Additional risk controls
required
(Step 5)
(Step 6)
Existing risk controls
C
Make solutions
Chemical Hazards
Exposure to:
Tris
EDTA
Dithiothreitol
Phenylmethanesulfonyl
fluoride (PMSF)
Benzamidine
Boric acid
Nonidet P40 (NP40)
Anneal Oligos
Physical Hazard
Heating block
Radioactive
labeling of Oligos
Exposure to radioactive
isotopes
Microcentrifuge
Tris: Irritant
EDTA: Toxic, irritant
Dithiothreitol: Irritant
Phenylmethanesulfonyl
fluoride (PMSF): Toxic,
corrosive
Benzamidine: Irritant
Boric acid: Irritant
Nonidet P40 (NP40):
Irritant
Heating block
3
D
M
2
E
L
3
E
M
(Apply the hierarchy of
risk controls)
C
L
R
Training
Irritant and toxic chemicals –
weigh out and make
solutions in fume hood
Standard PPE lab coat,
goggles and double gloves.
Electrical equipment testing
Burn hazard
Training
Radiation
Standard PPE lab coat,
goggles and double gloves.
Microcentrifuge
Electrical appliance –
electrocution hazard
R
(Step 7)
Standard PPE lab coat,
goggles and gloves.
Electrical appliance –
electrocution hazard
Radiation exposure,
potentially carcinogenic,
mutagenic and
teratogenic
L
Risk Rating with additional controls *
Radiation Protection Training
Use of shielding, time and
distance controls
Electrical equipment testing
Training
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Risk Assessment and Control Form
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 2.6, 16/07/2008
Making
acrylamidde Gel
Chemical Hazards
Exposure to:
Acrylamide: Toxic to
peripheral and central
nervous system,
carcinogenic, mutagenic,
developmental toxicity.
Severe exposure can
cause death. Avoid
ingestion, inhalation and
skin exposure.
Acylamide
Ammonium persulfate
(APS)
TEMED
APS: Irritant
Preparing EMSA
samples
Running samples
on gel
Exposure to radioactive
isotopes
Electrical appliances
Power pack
Chiller
Gel drying and Xray film exposure
Exposure to radioactive
isotopes
Electrical appliances
Gel dryer
Standard PPE lab coat,
goggles and gloves.
3
E
M
3
E
M
2
E
L
Radiation Protection Training
Use of shielding, time and
distance controls
Training
Standard PPE lab coat,
goggles and gloves.
Radiation exposure,
potentially carcinogenic,
mutagenic and
teratogenic
Radiation Protection Training
Electrical appliances:
electrocution
Electrical equipment testing
Electrical equipment
electrocution and burns
M
APS: Weigh out and make
solutions from powders in
fume hood
Radiation
Radiation exposure,
potentially carcinogenic,
mutagenic and
teratogenic
E
Training
TEMED: Buy as solution,
small volumes. Pipette in
fume hood
Radiation
4
Liquid handling in fume hood.
Wear double gloves.
TEMED: Toxic,
flammable
Radiation exposure,
potentially carcinogenic,
mutagenic and
teratogenic
Exposure to radioactive
isotopes
Standard PPE lab coat,
goggles and double gloves.
Use of shielding, time and
distance controls
Training
Standard PPE lab coat,
goggles and gloves.
Radiation Protection Training
Use of shielding, time and
distance controls
Electrical equipment testing.
Training
* C = consequence
L = likelihood
R = risk rating
from the UNSW Risk Rating Procedure
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Date Effective: 01/01/2007
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Current Version: 2.6, 16/07/2008
Step 8 Documentation and supervisor approval
Completed by:
S.Palmer
(signature)
Authorised by: Edna Hardeman
(signature)
Date: Jun09
Step 9: Implement the additional risk controls
identified
Indicate briefly what additional risk controls from Step 6 above were implemented, when and by whom.
Risk control:
Date:
Implemented by:
Risk control:
Date:
Implemented by:
Risk control:
Date:
Implemented by:
Risk control:
Date:
Implemented by:
Step 10: Monitor and review the risk controls
It is important to monitor risk controls and review risk assessments regularly. Review is required when there is a change in the process, relevant legal changes, and where a cause for concern has
arisen. Reviews could be scheduled on an annual basis. If the risk assessment has substantially changed a new risk assessment is warranted.
Review date:
Reviewed by:
Authorised by:
Review date:
Reviewed by:
Authorised by:
Review date:
Reviewed by:
Authorised by:
Review date:
Reviewed by:
Authorised by:
Documentation
It is a requirement that legal and advisory documentation that supports this risk assessment be listed. Such documentation includes Acts, Regulations, Australian Standards and Codes of Practice,
where applicable.
NSW OHS Act 2000
NSW OHS Regulation 2001
The NSW Radiation Control Act (1990) and Regulation (2003)
Code of Practice for the Labelling of Workplace Substances
AS/NZS 2243.2:2006. Safety in laboratories. Part 2: Chemical aspects
AS/NZS 2243.3: 2006 Safety in laboratories Part 3: Microbiological aspects and containment facilities
AS 2243.4-1998 Safety in laboratories - Ionizing radiations
AS/NZS 2243.6-1990. Safety in laboratories. Part 6: Mechanical Aspects.
AS/NZS 2243.3:2006 Safety in Laboratories Part 7 Electrical aspects
AS/NZS 2161.1:2000 Occupational Protective Gloves – Selection, Use and Maintenance
Safe Work Procedure Form (OHS026)
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Risk Assessment and Control Form
Date Effective: 01/01/2007
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Current Version: 2.6, 16/07/2008
UNSW Concise OHS Risk Rating Table
OHS697
What you need to do
1. Consider what can go wrong that can hurt someone
2. Determine what the most likely outcome would be - Consequences
3. Determine how likely those consequences are - Likelihood
4. Calculate the risk rating
5. Required action
How severely could someone be hurt
death or permanent disability to one or more persons
hospital admission required
medical treatment required
first aid required
injuries not requiring first aid
CONSEQUENCES:
Severe
Major
Moderate
Minor
Insignificant
How likely are those consequences?
expected to occur in most circumstances
will probably occur in most circumstances
could occur at some time
is not likely to occur in normal circumstances
may occur only in exceptional circumstances
LIKELIHOOD:
Almost certain
Likely
Possible
Unlikely
Rare
CONSEQUENCES
Insignificant
1
Minor
2
Moderate
3
Major
4
Severe
5
M
H
H
VH
VH
M
M
H
H
VH
Possible
C
L
M
H
H
VH
Unlikely
D
L
L
M
M
H
Rare
E
L
L
M
M
M
LIKELIHOOD
Almost
certain
A
Likely
B
Risk level
Very high
High
Medium
Low
Required action
Act immediately:
The proposed task or process activity must not proceed. Steps must be taken to lower the risk level to as
low as reasonably practicable using the hierarchy of risk controls.
Act today:
The proposed activity can only proceed, provided that:
(i) the risk level has been reduced to as low as reasonably practicable using the hierarchy of
risk controls;
(ii) the risk controls must include those identified in legislation, Australian Standards, Codes of
Practice etc.
(iii) the risk assessment has been reviewed and approved by the Supervisor and
(iv) a Safe Working Procedure or Safe Work Method has been prepared.
(v) The supervisor must review and document the effectiveness of the implemented risk
controls.
Act this week:
The proposed task or process can proceed, provided that:
(i) the risk level has been reduced to as low as reasonably practicable using the hierarchy of
risk controls;
(ii) the risk assessment has been reviewed and approved by the Supervisor and
(iii) a Safe Working Procedure or Safe Work Method has been prepared.
Act this month:
Managed by local documented routine procedures which must include application of the hierarchy of
controls.
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UNSW Concise OHS Risk Rating Table
Effective date: 01/01/2007
Uncontrolled document when printed
Current Version: 2.6,16/07/2008