CMB3001 Sample Protocol
Part B2: Sample protocol
Assay: Agarose gel electrophoresis of DNA
Protocol number: 1
Equipment
Agarose gel former and end stops
10-well gel comb
Electrophoresis tank
Power pack
Microwave oven
Weigh balance
250ml conical flask
2x500ml storage bottles
500ml measuring cylinder
30ml universal tube
Eppendorf tubes
Gilson pipettes and tips
UV transilluminator
Gel-doc digital imager
Carbon filter (for EtBr disposal)
Insulated gloves
Safety glasses
Protective dust mask (for weighing SDS)
UV protective face visor
Nitrile gloves
Reagents
Agarose
Tris HCl
Boric acid
EDTA
Glycerol
Bromophenol blue
Xylene cyanol
Ethidium bromide solution 1mg/ml (buy ready made from commercial supplier)
DNA molecular weight markers
Solutions
10xTBE (Tris-Borate-EDTA). Prepare 500ml in 500ml plastic storage bottle.
890mM TrisHCl, 890mM Boric acid, 20mM EDTA
Add the following to 400ml H2O:
54g Tris base.
27.5g Boric acid
4.6g EDTA
Adjust volume to 0.5L with additional distilled dH2O
1xTBE. Prepare 500ml in 500ml plastic storage bottle.
Add 50ml 10x TBE to 450ml dH2O for 1x TBE working stock.
DNA loading dye. Prepare 10ml in 30ml plastic universal tube.
Dissolve in 6.25 ml of H2O:
0.025g of Xylene cyanol
0.025g of Bromophenol Blue
Add:
1.25ml of 10% w/v SDS
CMB3001 Sample Protocol
12.5ml of glycerol
[10% w/v SDS = 10g SDS dissolved in 100ml H2O]
Method (step by step)
Preparing agarose gel
Weigh 1g agarose in small weigh boat. Transfer to 250ml conical flask and add
100ml 1x TBE.
Heat in microwave oven on medium heat for 2min or until just boiling. Remove from
oven wearing insulated gloves and eye protection. Swirl gently to mix. Repeat until
agarose is fully dissolved.
Allow to cool for 5min. While cooling insert end-blocks into gel former. Add 1ul
ethidium bromide solution (CARE - carcinogen). Swirl flask gently to mix
thoroughly.
Pour gel carefully into gel former and add gel comb to form wells. Allow to cool
until set.
Waste disposal
Dispose of gel by incineration (yellow bag).
Dispose of TBE buffer from tank (contains ethidium bromide) by passing through
carbon filter prior to disposal to drain (down sink).
Place glass and plasticware in metal boxes for washing and drying.
Risk Assessment
The following hazards are identified in connection with this protocol
Risk of explosion.
The agarose gel must be heated to boiling point in a microwave oven. In order to
avoid explosion the flask must not be fitted with a lid or stopper so that pressure is not
allowed to build up in the flask.
Risk of burning/scalding.
The agarose gel must be heated to boiling point in a microwave oven. Insulated
gloves and safety glasses must be worn when handling the hot flask.
Carcinogen risk.
Ethidium bromide is a potent carcinogen (see accompanying COSHH form). Nitrile
gloves must be worn at all times. To avoid risk of inhalation the ethidium bromide
must not be handled as a solid but will be bought from a commercial supplier as a
ready made solution. All liquid waste containing EtBr must be decontaminated by
passing through a carbon filter prior to disposal to drain. Solid agarose gels
containing EtBr must be disposed of by incineration (yellow bags). Gels must be
transported inside a plastic box to avoid the risk of dripping EtBr containing buffer on
the bench or floor.
UV risk.
Exposure to UV light presents a carcinogen risk A full UV-protective face visor
must be worn at all times when the transilluminator is switched on.
SDS; irritant risk (see accompanying COSHH form).
A protective dust mask and nitrile gloves must be worn when weighing out solid SDS.