Procedure:
School/Department:
SOP prepared by:
School of Biological Sciences
Joseph Dimauro & Nick Coleman (SMB) edit M Joseph
Section 1 - Personal Protective Equipment
1.
Wear a proper-fitting buttoned-up lab coat for entire procedure. Ensure closed in shoes are worn, and hair tied back.
2.
Wear proper fitting heat resistant gloves while preparing and pouring agarose.
3.
Wear disposable gloves (eg latex or nitrile) when handling the gel directly, and during the staining and photographic procedure.
4.
If photographing with a transilluminator, ensure UV-proof full face shield is worn, unless you are certain the instrument cannot expose the user to UV light. Most modern transilluminators have a safety override so that UV is switched off as soon as door is opened.
Section 2 – Potential Hazards + Safety precautions
1.
For a full list of hazards and precautions refer to Risk assessment for agarose gel electrophoresis.
2.
Briefly, the hazards involved are electric shock from the electrophoresis apparatus, burns from hot agarose, explosion in microwave if a sealed bottle is heated, and cancer risk from ethidium bromide.
Section 3 – Procedure
1.
Prepare agarose gel according to instructions, typically in 0.7-2% agarose and
1xTAE or 0.5 x TBE buffer. Ensure all glassware is clean. If you plan to excise bands from the gel, TAE must be used, since the borate in TBE can interfere with downstream enzymatic processes such as ligation.
2.
Prepare agarose using autoclave, stirrer hot plate or microwave, do not over boil. Ensure that the vessel containing the agarose has NO LID, or a VERY LOOSE-
FITTING LID. Do not walk away from the heating agarose, return to check at frequent intervals. If microwaving, it is safer to use a lower power for a longer time, rather than high power for a short time.
3.
Retrieve gel using heat-resistant gloves, allow agarose to cool to 50-60  C in waterbath. Pour gel and set up electrophoresis gear on a clear level bench in a well ventilated area.
4.
Stains such as GelRed or ethidium can be added directly to the gel, but keep in mind this will change how some DNAs run (esp. supercoiled circular DNAs). If you add ethidium to the gel, the whole apparatus will become contaminated and hazardous, and the buffer must be disposed of as hazardous chemical waste. In this case, the whole electrophoresis area and gel boxes must be labelled
“Ethidium contaminated”.
5.
When gel has set solid (20-30 min at room temp), remove well-formers and endformers (if used), and pour in running buffer (same as used for making gel, either
0.5 x TBE or 1 x TAE). Buffer should JUST cover the gel.
6.
Ensure the terminals are connected the correct way (red to red, black to black), and ensure your samples are at the negative end of the gel, running toward the positive end. Ensure the powerpack is in good working order, inspect leads for damage or exposed wiring. Do not use if damaged.
7.
Mix your DNA samples with loading buffer and load into the sample wells. PUT A
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LID ON THE GEL BOX. Run the gel at an appropriate voltage (depends on size of gel, small gels use approx 100 V, large gels use approx 200 V). Ensure current is flowing - milliamps on powerpack should be non-zero, and bubbles should be produced from electrodes. After a few minutes, check to see that blue indicator dye is running in the right direction and therefore polarity is correct.
8.
Once gel has run, transfer to stain (if no stain in gel), and allow 20-30 min to stain.
Can destain in water or buffer to enhance contrast (again 20-30 min), this is not essential.
9.
Put on gloves (eg. nitrile) and transfer gel into a plastic box with a tight fitting lid.
Take off gloves, go to room with transilluminator, put new gloves on, take gel out, and photograph on transilluminator. If you have a transilluinator within the same lab where the gel was run, there is no need for the plastic box, and the change of gloves.
10.
Once photographed, dispose of gel into hazardous waste bag.
11.
Appropriately wash all glassware AND APPARATUS. If you have used ethidium in the gel, the first few rounds of rinsate should go into liquid ethidium waste, not down the sink.
Section 4 – Disposal / Spills / Incidents
1.
All ethidium solution, wash, buffers and stained material to be disposed of as hazardous waste, appropriately labelled and taken to Hazardous Waste Bunker.
Glassware & Apparatus after initial rinse steps can be routinely washed.
2.
Spills need to be dealt with via the spills procedure and kit located in your area.
3.
Incidents are to be logged online via the University Incidents procedure and immediately notify your supervisor. Incidents would include exposure to ethidium, burns from hot agarose, electrical shock, or a large spill of ethidium containing solution (eg. > 1 L)
Section 5 – Repairs / Certification / Validation
1.
Ensure all apparatus is working correctly by testing and inspecting wiring and connections.
Section 6 – Relevant Material safety data sheets / References
1. These can be found as a hard copy in the associated laboratory or online using
the ChemAlert software. The relevant MSDS are those for ethidium bromide and
boric acid (or sodium borate).
Section 7 - References
1. Detailed protocols for gel electrophoreses can be found in many teaching practical manuals.
2. Risk assessment for agarose gel electrophoresis.
3. Risk assessment and SOP for Microwave Oven
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SOP Training Confirmation
By signing below, these individuals confirm that they have read and understood the SOP, and agree to always follow the instructions in this
SOP when performing this procedure.
Name Signature Date Position
Supervisor employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student
WHS Committee Approval
Representative: A. Prof Frank Seebacher Chair Safety Committee
Signature: ........................................................ Date: .....................................
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