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The Comet Assay is a single cell gel
electrophoresis
assay for evaluating DNA damage in cells
• Based on denatured, cleaved DNA fragments migrating out
of the cell when a current is applied whereas undamaged
DNA migrates slower
• Applications
• genotoxicity testing, human biomonitoring,
• cellular response to DNA damage, cancer risk assessment,
• cancer cell resistance to treatment
Assay Overview
1. Cells mixed with low melting
agarose at 37°C (LM Agarose)
2. Immobilize cells on
CometSlide™
3. Treat cells with Lysis Solution
(removes membranes and histones
from the DNA)
4. Samples treated with alkali
(unwinds and denatures DNA)
5. Alkaline electrophoresis
performed (reveals DNA breaks)
6. Samples stained with intercalating
dye and visualized by
epifluorescence microscopy.
SYBR® Green I
DNA migration out of nucleoid
Advantages
• Precoated slides to keep samples in place
• Avoids time consuming application of multiple
agarose layers
• No interference with fluorescent viewing and
analysis as seen with sand blasting
If agarose falls off slide
• Slides were washed
• Electrophoresis solution too hot
• Cells not washed to remove media
• Agarose percentage too low
• Agarose not fully set before processing samples
• Agarose unevenly set on slide
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