Sports and Exercise Sciences Research Institute
Standard Operating Procedure
Comet Assay
(SS/SOP0018)
Electrophoresis, used in this assay, is a chemical separation technique which
separates molecules (DNA, RNA and proteins) based on their size and electrical
charge
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Only trained personnel will be permitted to operate the electrophoresis
equipment and signed the COSHH Assessment Record Form that the
relevant COSHH forms have been read
Equipment is checked for leaks and/or damage prior to use by technical staff
Power supply is PAT tested annually
User must wear lab coat, heat protective and nitrile gloves and eye protection
Manufacturer’s build in safety precautions to minimize risks
SOLUTIONS REQUIRED
PROTOCOL
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Heat 1% normal melting point agarose in PBS to 80°C for 5 minutes
(depending on quantity of agarose); then cool to 44°C
Pre-coat glass slides with the 1% agarose above
Heat 1% low melting point agarose in PBS to 80°C for 5 minutes (depending
on quantity of agarose); then cool to 37°C
Mix the cells with 1% low melting point agarose above (50 µl of cells to 150 µl
LMPA), apply to slide and allow to solidify under a coverslip
The cells are lysed with lysis solution for at least 1 hour at 4°C
!!!!!!!!!!!!!!INCUBATION PERIOD WITH ENZYME!!!!!!!!!!!!!!
Place slides in the electrophoresis buffer and leave for 20 minutes at 4°C
Switch power supply on and set the voltage to 25 and the current to 300 mA
and leave for 20 – 30 minutes at 4°C.
Neutralise the slides with PBS firstly for 5 minutes at 4°C and then distilled
H2O for 5 minutes at 4°C. Allow the slides to dry overnight at room
temperature.
Stain with SYBR Gold or DAPI and gently roll on the see-saw shaker for 20 –
30 minutes.
Allow the slides to dry and quantify under the microscope.