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 Medicine
Research Unit
Faculty/Division: Medicine
Document number
SOMS.CGM.RA039
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:: Northern blotting
Step 1: Identify the activity
Describe the activity:
DEPC treatment in general
Add DEPC (Diethyl pyrocarbonate) to a final concentration of 0.1%. shake and leave at room temperature for at least 1hr
Autoclave for at least 15mins/L – this inactivates DEPC and generates EtOH and CO2
NB: Tris-containing solutions CANNOT be treated with DEPC and MOPS can be treated but will be turned yellow by autoclaving.
Solutions
RNA loading solution
glycerol (40%) and water – treat with DEPC.
20XSSC
88.23g Tri-sodium citrate
175.32g NaCl
Add 800mls H2O until dissolved
Adjust pH to 7-8
Make up to final volume of 1000ml
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10xMOPS
41.2g 3(N-morpholino) propanesulfonic acid (MOPS)
10.9g Sodium acetate, 3-hydrate
3.7g EDTA sodium salt
Add 800ml nuclease-free water
Adjust to pH7 with with NaOH (prepared in nuclease free water)
Make up to final volume of 1000ml
Filter sterilize
1M NaHPO4 pH 7.2
For 500 mL volume, dissolve in distilled water :
70.98g Na2HPO4
~2ml 85% H3PO4 (phosphoric acid)
Church (hybridization) Buffer
0.5M Na2HPO4 pH 7.2
(25ml of 1M stock)
1% BSA (fraction V)
(5ml of 10% stock)
7% SDS
(17.5ml of 20% stock)
1mM EDTA
(0.1ml of 0.1M stock)
2.4ml H2O
(up to 50ml total)
PROTOCOL – GEL PREPARATION AND ELECTROPHORESIS
1. Prepare the MOPS/Formaldehyde gel as follows: For 300mls of GEL Preheat 17.5 ml of Formaldehyde and 30 ml 10 x MOPS buffer at 55°C.
Dissolve 3–4.5 g of Agarose in 250 ml of nuclease free water. Cool to 55°C. Add the 10 x MOPS buffer and Formaldehyde, mix and pour immediately.
Allow the gel to set.
2. Prepare the RNA sample(s), using the table below (assuming 30ul total):
RNA
Formaldehyde
Formamide
10 x MOPS buffer
Water
TOTAL
Volume (l)
V
5.5
15
1.5
8-V
30
final conc
2.2 M
50%
0.5X
Place the sample(s) at 55°C for 15 minutes to denature. After denaturation add 3 μl of 10 x RNA loading solution. Mix and load onto the Agarose gel. Add
EtBR if required (see notes below).
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3. Separate the RNA samples using 1 x MOPS buffer as the electrophoresis buffer.
4. Following electrophoresis, visualize the RNA within the gel with UV light and photograph (ensure all skin exposed to UV is covered and face is
covered by a UV-opaque facemask).
5. Place the gel in a suitable tray or dish and cover with distilled water. Incubate the gel with gentle agitation for 15 minutes.
6. Discard the water and replace with sterile 10 x SSC. Agitate for 15 minutes. Repeat this step once more.
7. Set up the capillary blot as described on page 15.
NORTHERN BLOT PROTOCOL
1. Cut a sheet of membrane to an appropriate size. (I use Hybond XL or N+ - A neutral membrane is supposed to be best but I haven’t found this in
practice).
2. Half fill a tray or glass dish of a suitable size with the transfer buffer – 10XSSC. Make a platform and cover with a wick made from three sheets of blotting
paper saturated in transfer buffer.
3. Place the treated gel on the wick platform upside-down. Avoid trapping any air bubbles between the gel and the wick. Surround the gel with cling film to
prevent the transfer buffer being absorbed directly into the paper towels (optional).
4. Place the membrane on top of the gel. Avoid trapping any air bubbles.
5. Place three sheets of blotting paper cut to size and saturated in 10XSSC on top of the membrane. Avoid trapping any air bubbles.
6. Place a stack of absorbent towels on top of the Hybond blotting paper at least 5 cm high.
7. Finally, place a glass plate and a weight on top of the paper stack. Allow the transfer to proceed overnight. The weight should not exceed 750 g for a 20
x 20 cm gel.
8. After blotting, carefully dismantle the transfer apparatus. Before separating the gel and membrane, mark the membrane to allow identification of the
tracks with a pencil.
9. Fix the nucleic acid to the membrane by baking at 80°C for 2 hours or by using an optimized UV crosslinking procedure.
10. Blots may be used immediately. Blots must be thoroughly dried if storage is required.
NORTHERN HYBRIDIZATION PROTOCOL
Prehybridisation:
1.Preheat church buffer at 65°C.
2.Rinse the membrane briefly in 5xSSC then place it in hybridization bottle containing 15 ml (small) or 25 ml (large) of church buffer.
3.Prehybridise for 10mins- 2hours at 65°C in rotating oven
Radioactive labeling of probe:
This section outlines the labeling of 25ng DNA probe with [-32P]dCTP by random priming using Ambion DECAPrimeII kit. This procedure must be done in the radioactive
room (see Radioactivity Safe Work Practice and Risk Assessment). It is important to wear appropriate PPE and to be trained in the safe use of radioactive substance.
Prepare the following mixture:
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DNA template
25 ng
10x random primer solution
2.5 L
Water
Make up to 14 L
Mix and quickly spin down in microcentrifuge.
Denature the DNA sample by heating at 95-100°C for 5 minutes in heating block. Chill on ice. Spin down quickly.
Add the following components in the same tube:
5x reaction buffer (containing all
5 L
dNTPs except dCTP)
Exonuclease-free Klenow enzyme
1 L
[-32P]dCTP
5 L
Flick the tube and quick spin.
Incubate at 37°C for 10 minutes.
Add 25 L 1xTE buffer into the tube, quick spin.
Removal of unincorporated nucleotides is achieved by exclusion chromatography using Sephadex G50 columns.
Cut the lid off an eppendorf tube, place a Sephadex column in the tube after shaking to resuspend the matrix and snapping off the cap.
Spin at 3000rpm for 1min
Put the column in a fresh screw-cap tube, add the 50 l of probe sample onto the column.
Spin at 3000 rpm for 2 minutes. Dispose of the radioactive column appropriately in a perspex waste box.
Hybridisation:
Heat the probe at 100oC to denature the probe
Add denatured probe to prehybridisation solution in the bottle. Ensure that radioactive probe does not touch the filter directly.
Incubate overnight at 65°C
Wash:
Carefully dispose of the radioactive hybridization solution into a liquid waste container that is placed in a perspex box.
Wash membrane twice with at least 250 mls 0.1xSSC + 0.1%SDS at room room temperature to remove the bulk of the probe. Dispose of wash solutions in the radioactive
waste.
Wash membrane twice with at least 250 mls 0.1xSSC + 0.1%SDS at 65 oC – high stringency wash. Dispose of wash solutions in the radioactive waste.
Detection:
Remove most of the excess wash solution by quick touch onto a 3MM paper. Wrap moist blot in plastic wrap, sealed well, before placing into a cassette.
In the dark room: expose membrane to X-ray film with an intensifying screen.
Store at -80°C for 1 to 6 days.
Develop the film (see Working with X-ray developer SWP and Risk Assessment)
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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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Tasks
Hazards
Associated risks
(Step 3)
(Step 4)
Risk rating with
existing controls *
Additional risk controls
required
Risk Rating with
additional controls *
(Step 5)
(Step 6)
(Step 7)
Existing risk controls
C
Make solutions
Chemical Hazards
Exposure to:
DEPC
MOPS
Phosphoric acid
SDS
Sodium Hydroxide
Physical Hazards
Use of electric stirrer
pH Meter
DEPC
Harmful, irritant,
combustible liquid
MOPS
Highly irritant on eye
contact
Phosphoric acid
Corrosive in contact with
skin and eyes or
ingestion
SDS
(Powder) Flammable,
irritant to skin and by
inhalation (severe).
3
L
D
R
(Apply the hierarchy of
risk controls)
C
L
R
M
Standard PPE lab coat,
goggles and gloves.
Training
Irritant and toxic chemicals –
weigh out and make
solutions in fume hood
SDS is much safer as a
20% solution. Buy in this
form
Electrical safety testing
Ethidium Bromide
Poison, may cause
heritable genetic
damage.
Sodium Hydroxide
Corrosive: will cause
severe chemical
burns/irritation in contact
with skin, eyes or by
ingestion . Dissolving
NaOH in water is
exothermic
Physical Hazards
Electrocution
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Make gel, prepare
RNA samples,
load and run gel,
Photograph under
UV light
Chemical Hazards
Exposure to:
Formaldehyde
Formamide
Ethidium Bromide
Formaldehyde
Poison, suspected
carcinogen, irritant
(especially by inhalation),
flammable.
Standard PPE lab coat,
goggles and double gloves.
Physical hazards
Power pack
Heating block
UV Radiation burn
Formamide
Irritant on skin, eyes or
ingestion
Formaldehyde must be
decanted in fume hood and
the agarose gel must be
poured in the fume hood.
Once the gel has cooled and
set, the fumes are minimal. It
is OK to run the gel outside
of the fume hood.
UV transilluminator
Use Gel-Doc contained
UV source and
photography unit
Northern Blotting
Chemical hazards
Exposure to
Formaldehyde
Ethidium bromide
Physical hazards
Power pack
Heating bloc
Radioactive
labeling of probes
and membrane
hybridization
Exposure to radioactive
isotopes
Physical hazards
Microcentrifuge
Hybridization oven
Heating block
UV stratalinker
Formaldehyde
Poison, suspected
carcinogen, irritant
(especially by inhalation),
flammable.
Ethidium Bromide
Poison, may cause
heritable genetic damage
Radiation
Radiation exposure,
potentially carcinogenic,
mutagenic and
teratogenic
Microcentrifuge
Hybridization oven
Heating block
UV stratalinker
3
D
M
2
D
L
3
D
M
Electrical equipment testing
Training
Ethidium Bromide is less
likely to be inhaled as a
solution. Buy as a 10mg/ml
solution. Handle and dispose
of with care wearing PPE.
Standard PPE lab coat,
goggles and double gloves.
Training
Washing of the gel reduces
formaldehyde fumes and
residual Ethidium bromide
concentration is tiny.
Standard PPE lab coat,
goggles and double gloves.
Radiation Protection Training
Use of shielding, time and
distance controls
Electrical equipment testing
Training
Electrical appliances –
electrocution hazard
* C = consequence
L = likelihood
R = risk rating
from the UNSW Risk Rating Procedure
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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:
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:
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.


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
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


NSW OHS Act 2000
NSW OHS Regulation 2001
The NSW Radiation Control Act (1990) and Regulation (2003)
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
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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
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Current Version: 2.6,16/07/2008