Faculty/Division: Medicine
Document number
SOMS.CGM.RA059
Initial Issue date
30/06/09
Current version
1.0
School/Unit: School of Medical Science, Neuromuscular and Regenerative Medicine
Research Unit
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:: Yeast 2-Hybrid analysis
Step 1: Identify the activity
Describe the activity:
Day 1
Inoculate the yeast strain into 5 ml of liquid medium (2x YPD or SD selection medium) and incubate overnight on a rotary shaker at 200 rpm and 30°C. Place a bottle of double strength
YPD broth (2x YPD) and a 250 ml culture flask in the incubator as well.
Day 2
1.
Determine the titer of the yeast culture by pipetting 10 ml of cells into 1.0 ml of water in a spectrophotometer cuvette and measuring the OD at 600 nm. Calculate the cell titer
2.
Transfer 50 ml of the pre-warmed 2x YPD to the pre-warmed culture flask and add 2.5 x 10 8 cells to give 5 x 10 6 cells/ml.
3.
Incubate the flask on a rotary or reciprocating shaker at 30°C and 200 rpm
4.
After about 4 hours, harvest the cells by centrifugation at 3000 g for 5 min, wash the cells in 25 ml of sterile water and resuspend in 1 ml of sterile water.
5.
Heat a 1.0 ml sample of salmon sperm carrier DNA for 5 min @ 100 o C and chill in an ice/water bath while harvesting the cells.
6.
Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge for 30 sec and discard the supernatant.
6.
Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge for 30 sec and discard the supernatant.
7. Add water to a final volume of 1.0 ml and vortex mix vigorously to resuspend the cells.
8.
Pipette 100 µl samples (ca. 10 8 cells) into 1.5 ml microfuge tubes, one for each transformation, centrifuge at top speed for 30 sec and remove the supernatant.
9.
Make up sufficient Transformation Mix for the planned number of transformations plus one extra. Keep the Transformation Mix in ice/water.
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Risk Assessment and Control Form
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Date Effective: 01/01/2007
Current Version: 2.6, 16/07/2008

Number of Transformations
Reagents
PEG 3500 50% w/v
1
240 µl
5 (6X)
1440 µl
10 (11X)
2640 µl
LiAc 1.0 M
Boiled SS-carrier DNA
Plasmid DNA plus Water
36 µl
50 µl
34 µl
216 µl
300 µl
204 µl
396 µl
550 µl
374 µl
Total 360 µl 2160 µl 3960 µl
10. Add 360 µl of Transformation Mix to each transformation tube and resuspend the cells by vortex mixing vigorously.
11. Incubate the tubes in a 42°C water bath for 40 min.
12. Microcentrifuge at top speed for 30 sec and remove the Transformation Mix with a micropipettor.
13. Pipette 1.0 ml of sterile water into each tube; stir the pellet by with a micropipette tip and vortex .
14. Plate appropriate dilutions of the cell suspension onto SC selection medium. For transformation with an integrating plasmid (YIp), linear construct or oligonucleotide, plate 200 µl onto each of 5 plates; for a YEp, YRp or YCp library plasmid dilute 10 µl of the suspension into 1.0 ml of water and plate 10 and 100 µl samples onto two plates each. The 10 µl samples should be pipetted directly into 100 µl puddles of sterile water on the SC selection medium.
15. Incubate the plates at 30°C for 3 to 4 days and count the number of transformants.
YPD medium
20 g/L Difco peptone
10 g/L Yeast extract
20 g/L Agar (for plates only)
• [Optional] For adenine-supplemented YPD (YPDA or YPAD), add 15 ml of a 0.2% adenine hemisulfate solution per liter of medium (final concentration is 0.003%, in addition to the trace amount of Ade that is naturally present in YPD). Adenine hemisulfate tolerates autoclaving.
Add H2O to 950 ml. Adjust the pH to 6.5 if necessary, then autoclave. Allow medium to cool to ~ 55°C and then add dextrose (glucose) to 2% (50 ml of a sterile 40% stock solution). Adjust the final volume to 1 L if necessary.
SD medium
6.7 g Yeast nitrogen base without amino acids
20 g Agar (for plates only)
850 ml H2O
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Date Effective: 01/01/2007
Current Version: 2.6, 16/07/2008

100 ml of the appropriate sterile 10X Dropout Solution
• Adjust the pH to 5.8 if necessary, and autoclave. Allow medium to cool to ~ 55°C before adding 3-AT, cycloheximide, additional adenine, or X-gal (see below).
• Add the appropriate sterile carbon source, usually dextrose (glucose) to 2%, unless specified otherwise for your application. Adjust the final volume to 1 L if necessary.
Describe the location:
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 exposed to heating block at 100 o C or near Bunsen flame.
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.
Tasks
Hazards
(Step 3)
Associated risks
(Step 4)
Existing risk controls
C
Risk rating with existing controls *
(Step 5)
L R
Additional risk controls required
(Step 6)
(Apply the hierarchy of risk controls)
Risk Rating with additional controls *
(Step 7)
C L R
Chemical Hazards
Exposure to:
Lithium Acetate 1M
3-amino-1,2,4-triazole
Lithium Acetate 1M –
Slightly hazardous in case of skin/eye contact
(irritant) or inhalation/ingestion
3-amino-1,2,4-triazole -
Slightly hazardous in case of skin/eye contact
(irritant) or inhalation/ingestion
Standard PPE lab coat, goggles and gloves.
Training
Irritant and toxic chemicals – weigh out and make solutions in fume hood
3 D M
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Risk Assessment and Control Form
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Date Effective: 01/01/2007
Current Version: 2.6, 16/07/2008

Physical Hazard
Shaking Incubator
Benchtop centrifuge
Spectrophotometer
Electrical appliance
– electrocution hazard
2 E L
T
o
Physical Hazard
Electrical equipment
Physical Hazard
Electrical equipment
Physical Hazard
Electrical equipment
Chemical Hazards
Exposure to:
Lithium Acetate 1M
Physical Hazard
Electrical equipment
Glass pipettes bent in
Bunsen flame
Electrocution
Burns
Electrocution
Rotor spin injury
Electrocution
Lithium Acetate 1M –
Slightly hazardous in case of skin/eye contact
(irritant) or inhalation/ingestion
Electrocution
Rotor spin injury
Burns from Bunsen
Stick injury from glass pipettes
* C = consequence L = likelihood
Standard PPE lab coat, goggles and gloves.
Electrical equipment testing
Training
Standard PPE lab coat, goggles and double gloves.
Training
Standard PPE lab coat, goggles and gloves.
Training
Standard PPE lab coat, goggles and gloves.
Electrical equipment testing
Training
R = risk rating
2 E
2 E
2 E
2 E
L
L
L
L from the UNSW Risk Rating Procedure
Standard PPE lab coat, goggles and gloves.
Electrical equipment testing
Training
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Risk Assessment and Control Form
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Date Effective: 01/01/2007
Current Version: 2.6, 16/07/2008

Step 8 Documentation and supervisor approval
Completed by: (name) S.Palmer (signature) Authorised by: (name)
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:
Risk control:
Risk control:
Date:
Date:
Date:
(signature)
Implemented by:
Implemented by:
Implemented by:
Date:
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
Code of Practice for the Labelling of Workplace Substances
AS/NZS 2243.2:2006. Safety in laboratories. Part 2: Chemical aspects
AS/NZS 2243.3:2002 Safety in laboratories Part 3: Microbiological aspects and containment facilities
Australian Standard AS2243.6-1990. Safety in laboratories. Part 6: Mechanical Aspects.
Australian Standard AS2243.7-1991. Safety in laboratories. Part 7: Electrical Aspects.
AS/NZS 2161.1:2000 Occupational Protective Gloves – Selection, Use and Maintenance
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Risk Assessment and Control Form
Uncontrolled document when printed
Date Effective: 01/01/2007
Current Version: 2.6, 16/07/2008

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
CONSEQUENCES:
Severe
Major
Moderate
Minor
Insignificant
LIKELIHOOD:
Almost certain
Likely
Possible
Unlikely
Rare
LIKELIHOOD
Almost certain
A
Likely
B
Possible
C
Unlikely
D
Rare
E
Risk level
Very high
High
Medium
Low
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
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
Insignificant
1
M
M
L
L
L
Minor
2
H
M
M
L
L
CONSEQUENCES
Moderate
3
H
H
H
M
M
Major
4
VH
H
H
M
M
Severe
5
VH
VH
VH
H
M
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