ohsrmPART B School of Molecular Bioscience.

HAZARDOUS PROCEDURE: NORTHERN BLOTTING & HYBRIDISATION.

Step 3. Assess the risk

Ref. # / Description of the hazard or hazardous job / Priority / Identification Date
1 / Ethidium bromide use in RNA sample preparation and visualization. Potential for direct contact with skin and eyes with harmful effects. Ethidium Bromide is a potent mutagen. / 4 / 21/04/2010
2 / Heating agarose solutions: Superheated solution overflowing from container can potentially cause significant burn injuries to eyes and to skin on arms, upper body and face. / 4 / 21/04/2010
3 / Formaldehyde use in gel casting and gel electrophoresis and RNA sample preparation. Potential for the inhalation of toxic fumes and direct contact with eyes and skin. May result in burns and permanent tissue damage. Formaldehyde is a confirmed human carcinogen (IARC-G1) and has acute toxic effects. / 3 / 21/04/2010
4 / Formamide use in RNA sample preparation. Potential for the inhalation of toxic vapours and direct contact with eyes and skin. May cause poisoning, severe burns and blistering. Symptoms of exposure may not be immediately painful or visible. / 4 / 21/04/2010
5 / RNA Electrophoresis. Potential for serious electrical shock or electrocution due to leaking chamber, faulty or corroded electrode cables, or faulty power supply. / 5 / 21/04/2010
6 / UV light use in DNA visualisation, and DNA-Membrane Fixing. Potential for UV-C exposure to skin and eyes. May cause tissue burns after a few seconds. May cause blindness upon prolonged exposure to eyes. Symptoms of exposure may not be immediately painful or visible. / 5 / 21/04/2010
7 / Preparing SDS (sodium dodecyl sulphate) solution. Potential for inhalation, which may cause pulmonary sensitization, or direct contact with eyes and skin, which may cause pain and redness. / 5 / 21/04/2010
8 / 32P radioisotope use in probing and washing northern membranes. Potential for radiation exposure. May cause tissue damage or radiation poisoning. (see Risk Assessment for radioactive isotopes) / 5 / 21/04/2010

What makes it hazardous? Consult with the workers to find out which factors are relevant:

Tick relevant boxes and record observations or comments.
 The nature of the hazards themselves. / This hazardous procedure is commonly used in RNA analysis. Failure to observe safety measures exposes the user, and potentially other work colleagues, to significant risk of physical injury, including tissue burns, eye injury, and electric shock, as well as toxic and radiation poisoning,
 The individual(s) involved / Requires specific training by authorised staff in the safe use of the above listed hazardous chemicals and equipment.
Record the names of those consulted when assessing the risk / Date
Nick Coleman, Peter Kerr, Joe Dimauro, Jenny Phuyal, Zia Ahmad, Angela Nikolic, Robert Czolij. / 31/03/2010

Step 4. Control the risk(s)

Control the risks(s) by addressing the risk factors found in Step 3.

Consider the hierarchy of hazard control and record what controls will be used in the short term and longer term. Record also who is responsible for implementing the control(s) and the due by date(s).

Describe the risk control(s) / Who is responsible for implementation / Due by date
ALWAYS wear personal protective equipment (PPE) throughout this procedure, including: lab coat, Nitrile gloves (except for heating agarose solutions, use heat insulating gloves instead), and safety glasses/goggles / Individual user and their supervisor / 21/04/2010
Using ethidium bromide:ONLY purchase and use pre-made ethidium bromide solutions - this removes the significant hazard of inhaling the toxic powder form of ethidium bromide.
LIMIT ethidium bromide work to designated areas.
USE barrier/filter tips when aliquoting ethidium bromide to help prevent contamination of pipettes.
Solutions containing ethidium must be disposed of as hazardous chemical wastes to room 225, not down the sink / Individual user and their supervisor / 21/04/2010
Heating agarose solutions:(See also MICROWAVE OVEN risk assessment)
ALWAYS wear thick heat-resistant gloves as part of your PPE.
NEVER exceed one-third the volume of a glass flask or bottle with your agarose solution. NB: If you choose to use a lid ensure it is unscrewed to allow sufficient ventilation – no lid is a safer option, as loose lids can become stuck on during the heating process.
DON’T heat solution on greater than 50% power.
ALWAYS swirl solution occasionally and gently while it is heating to prevent superheated overflow.
NEVER leave the heating of your agarose solution unattended. / Individual user and their supervisor / 21/04/2010
Formaldehyde
ALWAYS work with formaldehyde in the fume hood to avoid inhaling toxic fumes. This includes aliquoting formaldehyde into RNA sample tubes, adding formaldehyde to your warm agarose solution before pouring, and when you perform gel electrophoresis.
USE barrier/filter tips when aliquoting formaldehyde to help prevent contamination of pipettes.
Solutions containing significant amounts of formaldehyde must be disposed of as hazardous chemical wastes to room 225, not down the sink.
Gels should be washed in a closed container to remove formaldehyde (and formamide) before handling them outside of the fume hood (eg. for photography of gel by GelDoc)
Dry northern gels in a container in the fume hood to remove all volatiles (formaldehyde & formamide) before discarding them through the ethidium bromide waste stream. / Individual user and their supervisor / 21/04/2010
Formamide
ALWAYS work with formamide in the fume hood. This includes aliquoting small volumes of formamide to RNA sample tubes.
USE barrier/filter tips when aliquoting formamide to help prevent contamination of pipettes.
Solutions containing formamide must be disposed of as hazardous chemical wastes to room 225, not down the sink / Individual user and their supervisor / 21/04/2010
RNA electrophoresis(Also see agarose gel risk assessment)
Inspect electrodes and cables for defects prior to use. DON’T use any electrophoresis equipment that you suspect has an electrical fault or is damaged – in this case, report item to supervisor/ lab manager/ safety officer for electrical testing, repair or replacement.
Ensure area around electrophoresis chamber is free of spills or leaks PRIOR and DURING the running of your gel.
Ensure the electrophoresis chamber has a secure LID which prevents accidental contact with the electrified buffer solution in the tank / Individual user and their supervisor / 21/04/2010
Ultraviolet light (Transilluminator)
If using an open transilluminator, WEAR UV-PROOF PERSPEX FACE SHIELD! If using Gel-Doc, keep doors of GelDoc closed when UV is in use. Also refer to Risk assessment / SOP for Ultraviolet Light Sources. / Individual user and their supervisor / 21/04/2010
SDS
Use P1 type respirator (protects against dusts and mists) when weighing out SDS powder – this is strongly irritating if inhaled. / Individual user and their supervisor / 21/04/2010
32P isotope
WEAR ALL APPROPRIATE PROTECTIVE CLOTHING (above), work in approved registered radiation lab (eg. Room 647), complete radiation safety training course before starting work, use perspex shield between radioactive source and body, wear personal dosimeter, check self and work area with geiger counter to ensure no spills and/or proper cleanup procedure. Also refer to Risk Assessment / SOP for Radioactive isotopes. / Individual user and their supervisor / 21/04/2010
CLEAN up all spills as per Material Safety Data Sheet (MSDS) recommendations.REPORTlarge-scale spills or contamination to supervisor and/or safety officer.DISPOSE of hazardous waste appropriately (rm 225). / Individual user and their supervisor / 21/04/2010
Proper training of staff / students is essential prior to performing Northern blots / Supervisor of researcher / 21/04/2010
Development and updating of Standard Operating Procedure (SOP) / SMB Safety Committee / 21/04/2010
Consulting SOP before performing this hazardous procedure, and observe all risk control recommendations. / Individual user and their supervisor. / 21/04/2010
Refer to MSDS or consult safety officer if further information is required. / Individual user and their supervisor. / 21/04/2010
Record the names of those consulted when deciding on risk control measures
Nick Coleman, Peter Kerr, Joe Dimauro, Jenny Phuyal, Zia Ahmad, Angela Nikolic, Robert Czolij. / 31/03/2010
PART B completed by: / Craig JACKSON / Date: / 31/03/2010