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Tech Tip: Single Cell Gel Electrophoresis (Comet) Assay

Overview
The Single Cell Gel Electrophoresis assay (SCGE), or Comet Assay, measures DNA damage from
individual cells based on the migration of denatured DNA through an electrophoretic field(1,2) (Fig. 1).
Individual cells or nuclei are embedded in agarose, lysed to expose their DNA, alkali treated to denature/
relax the DNA, and electrophoresed to separate the DNA.(3,4) The DNA is visualized by EtBr, silver stain,
or fluorescent dyes. Damaged DNA containing strand breaks migrates farther in the gel than intact DNA,
creating an image resembling a celestial comet. When performed under alkaline conditions, SCGE detects
(but does not distinguish between) double-stranded breaks, single-stranded breaks, alkali-labile sites
(single-stranded breaks), incomplete excision repair (single-stranded breaks), DNA-DNA interactions, and
DNA-protein interactions. The specificity of SCGE can be enhanced to investigate specific types of DNA
damage by adding DNA modifying enzymes. Enzymes which have successfully been used in the comet
assay include Mut M (Fpg) for oxidative damage, AlkA for alkylation damage, Endo III for oxidized
pyrimidines, and Proteinase K for DNA-protein interactions (see Table I: Enzymes to Study DNA).

Control

Fig. 1. Single Cell Gel Electrophoresis

performed on Mouse NIH/3T3 cells
incubated with Mut M (Fpg) under
Control and severe DNA damage (5%
hydrogen peroxide) conditions.

H2O2

EtBr SYBER® Green I

The genotoxic effects of virtually any chemical or industrial compound can be investigated using
SCGE. Analyses can include any organism, tissue, or culture from which a single cell or nuclei
suspension can be isolated. Resulting DNA comets are analyzed visually by estimating the extent of
DNA damage or measuring tail length. Image analysis tools are available to analyze various
parameters of the comets including tail moment, percent migrated DNA, and tail length (for more
information, see Ref. 9). USB offers reliable nuclease-free reagents to maximize reproducibility and
accuracy of the SCGE assay as well as various DNA repair, recombination, and replication enzymes to
extend the utility of the SCGE assay (see Table II).
Table I. Enzymes to Study DNA:

Table II. Ultrapure Reagents from USB for SCGE:

Product No.
Agarose - Separation ≥ 500 bp, Genetic PerformanceCertified™ 75817
PBS, 10X Solution, pH 7.4 75889
Agarose - Low Melt, Separation ≤1000 bp, Genetic Performance Certified™ 32829
Triton® X-100 22686
EDTA, 0.5M Solution 15694
TBE Buffer, 5X Solution 75891
TE Buffer, 1X Solution 75893
TE Buffer, 50X Solution 75834
Tris-HCl, 1M Solution, pH 7.5 22639
Ethidium Bromide Drops 75816
Sodium Chloride, 5M Solution 75888
Tris 75825
Water, Nuclease-Free 71786

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Buffer Preparation:
Lysis Solution:
For 1 L
2.5M NaCl 500 ml 5M NaCl (PN 75888)
0.1M EDTA 200 ml 0.5M EDTA, pH 8.0 (PN 15694)
10mM Tris-HCl, pH 10 1.2 gm/L Tris (PN 75825)
Adjust pH to 10.0 with NaOH pellets.
H2O (PN 71786) to 1 L
Filter through a 0.5 µm filter. Chill to 4°C for convenience in assay.
Before use add Triton X-100 (PN 22686) to 1% and DMSO to 10% (optional). Addition of Triton X-100
will turn solution slightly cloudy. DMSO prevents oxidation during lysis incubation.
Denaturation Solution (make fresh daily):
For 1 L
300mM NaOH 12 gm NaOH
1mM EDTA 2 ml 0.5M EDTA (PN 15694)
H2O (PN 71786) to 1 L
pH should be >13
Exposure of cells to genotoxic substances:
Genotoxic substances should be treated with extreme care. Cells can be exposed while in culture by
adding directly to the growth media or while embedded in agarose on the slide during the SCGE assay.
Chemicals, preferably dissolved in PBS, can be applied to the slides by adding 50 µl of solution per
section (half) of the microscope slide and dispersing with a cover slip, or alternatively slides can be
immersed in the chemical solution (e.g. hydrogen peroxide). Incubate on ice to inhibit the endogenous
DNA repair enzymes.

Single Cell Gel Electrophoresis Assay Protocol

Pre-coating of Glass Microscope Slides:
1. Dip a glass microscope slide into 1.5% molten agarose (PN 75817) made up in distilled/filtered
water. Immediately wipe all agarose off the back of the slide.
2. Place slide on bench top or in drying oven and allow the agarose to dry completely. Slides can be
used for up to 1 week when stored at RT.
Embedding of Cells:
3. Centrifuge cells (e.g. cells grown in culture or trypsinized cells from plates) at 2000 x g in a
microcentrifuge for 1 min at 4°C, discard the supernatant.
4. Resuspend cells in 1 ml of 1X PBS (10X PBS, PN 75889).
5. Centrifuge cells at 100 x g in a microcentrifuge for 5 min at 4°C, discard the supernatant.
6. Resuspend cells in PBS to about 25000 cells per ml.
7. Mix 5 µl of cell suspension (about 125 cells) with 35 µl 0.8% Agarose, Low Melt (PN 32829) (0.8%
LM agarose can be microwaved to liquefy and stored at 37°C until use). Pipette mixture
immediately onto half a microscope slide and cover with a 22 x 22 mm cover slip.
8. Repeat application of cells for second half of slide.
9. Place slides on ice or at 4°C for 5 min to allow agarose to form gel.
10. Remove cover slips.
11. Cells are now accessible for additional manipulation (e.g. exposure to UV irradiation or to chemical
agents).
Lysis of Cells:
12. Cover slides in staining trays with ice cold Lysis Solution. (Note: Triton X-100 lyses the cells. High
salt removes histones from DNA.)
13. Incubate on slow shaker for 1 hr at 4°C. Lysis times may vary depending on the cells being used.
14. Rinse slides with filtered water.
15. Wash slides in filtered water 3 times for 5 min each wash.

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Treatment of Slides with DNA Modifying Enzymes (Optional, Mut M example):
16. Dilute Mut M (Fpg) 1 x 102 to 103 in reaction buffer (PN 71430).
17. Add 50 µl of enzyme solution per half of microscope slide. Cover with a cover slip.
18. Incubate at 37°C for 30 min.
19. Remove cover slip.
Denaturation/DNA Unwinding:
20. Cover slides with Denaturation Solution.
21. Incubate with slow shaking for 20 min at 4°C.
Electrophoresis:
22. Pre-equilibrate slides by covering with 1X TBE (5X TBE, PN 75891) buffer for 10 min. Place in
horizontal submarine gel electrophoresis unit.
23. Electrophorese samples in 1X TBE. For a 13 cm electrode distance, electrophorese for 3-1/2 min at
25V, 3mA. Vary time of electrophoresis as appropriate.
24. Wash slides in filtered water 2 times for 5 min each wash.
25. Slides can be stained immediately or dried completely for later use. For drying, slides can be
dipped in 100% EtOH (or MeOH) and dried at RT or 37°C.
Electrophoresis Alternative: Slides can be electrophoresed in fresh Denaturing Solution at 25 volts
for 10 min for ss and ds DNA breaks. Electrophoresis time may vary. Neutralize in 0.4M Tris, pH 7.5
(1M Tris, pH 7.5, PN 22639)
Visualization of DNA:
SYBR® Green I Nucleic Acid Stain - Dilute SYBR Green I stock 1:10,000 in TE Buffer (pH 7.5-8.0) as
directed by manufacturer. Pipet 100 µl diluted SYBR Green I onto dry slides and add cover slips.
Visualize under fluorescence microscope.
Ethidium Bromide - Pipette 80 µl (2 drops) of 2 µg/ml EtBr (PN 75816) onto dry slide and apply cover
slip. Incubate for 30 min at room temperature. Remove cover slips and dip slides in filtered H2O to
rinse. Visualize under fluorescence microscope.
Silver Stain - Silver stained slides have the advantage of visualization on a light microscope and slides
can be stored for future reference. For detailed protocol, see reference 10.

Troubleshooting:
No comets on Control or Experimental slides.
1. Cells not lysed.
- Extend incubation time in lysis buffer.
2. Lysis buffer not effective.
- Test alternative lysis buffers.
3. DNA not electrophoresed.
- Increase voltage.
- Increase electrophoresis time.
4. DNA not fully denatured.
- Increase denaturing time.
All cells, Control and Experimental, give comets.
1. DNA damage.
- Use chilled buffers, substitute Hank’s Balanced Salt Solution (HBSS) for PBS
- Shorten time between cell harvest and start of assay.
- Reduce cell exposure to fluorescent light in work area.
- 0.8% LM agarose was too hot, cool to 37°C.
2. Electrophoretic field too high.
- Reduce electrophoretic time.
- Reduce voltage.

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Agarose lifting off of slide.
1. Too much agitation during washes/incubation steps.
- Reduce agitation rate of shaker.
- Perform washes and incubations on the bench top without agitation.
2. Hydrated for too long.
- Dry slides completely before staining DNA (after electrophoresis).
Comets on a slide vary widely in tail length and orientation of tail.
1. Current through the gel was inconsistent because the gel layer was not uniform.
- Use cover slips when dispensing the agarose + cells onto the slide to create a uniform gel layer.
Comets near edges of gel layer do not match comets on rest of slide.
An ‘edge effect’ in the assay. Do not score comets along edge of gel layer.

Plant Nuclei Comet Assay Procedure(11):

Additional Supplies:
10% SDS - PN 77504
1. Treat leaves with UV, H2O2 or other.
2. Wash 4 small leaves in distilled water.
3. Place leaves in a petri dish with 3 ml cold PBS + 1 mM EDTA, slice the leaves with a razor blade
into small pieces.
4. Combine 15 µl of leaf extract with 100 µl 0.8% Agarose, Low Melt, mix, pipette onto half a pre-
coated microscope slide (See “Precoating of Glass Microscope Slides” in above protocol). Cover
with cover slip immediately.
Note: Amount of leaf extract suspended in agarose can be varied to alter density of comets on the
slide.
5. Repeat for second half of slide.
6. Place on ice for 5 min to solidify.
7. Remove cover slips.
8. Cover slides in Plant Lysis Solution (2.5% SDS in TBE Buffer) chilled to 4°C.
9. Incubate on slow shaker for 1 hr at 4°C.
10. Rinse slides with filtered water.
11. Wash the slides in filtered water for 10 min. Repeat rinse and wash steps.
12. Follow “Treatment of Slides with DNA Modifying Enzyme”, “Denaturation/DNA Unwinding”,
“Electrophoresis”, “Electrophoresis Alternative”, and “Visualization of DNA” as stated for cell lines.

References
1. OSTLING, O., AND JOHANSON, K. J. (1984) Biochemical and Biophysical Research Communications 12, 291-298.
2. SINGH, N. P., MCCOY, M. T., TICE, R. R., AND SCHNEIDER E. L. (1988) Experimental Cell Research 175, 184-191.
3. TICE, R. R., AGURELL, E., ANDERSON, D., BURLINSON, B., HARTMANN, A., KOBAYASHI, H., MIYAMAE, Y., ROJAS, E.,
RYU, J. C., AND SASAKI Y. F. (2000) Environmental and Molecular Mutagenesis 35, 206-221.
4. HARTMANN, A., AGURELL, E., BEEVERS, C., BRENDLER-SCHWAAB, S., BURLINSON, B., CLAY, P., COLLINS, A., SMITH, A.,
SPEIT, G., THYBAUD, V., AND TICE, R. R. (2003) Mutagenesis 18, 45-51.
5. WOZNIAK, K. AND BLASIAK J. (2003) Mutation Research 535, 127-139.
6. SPEIT, G., SCHÜTZ, P., BONZHEIM, I., TRENZ, K., AND HOFFMANN, H. (2004) Toxicology Letters 146, 151-158.
7. COLLINS, A. R., DUSINSKÁ, M., AND HORSKÁ, A. (2001) Acta Biochimica Polonica 48, 611-614.
8. COLLINS, A. R., DOBSON, V. L., DUSINSKA, M., KENNEDY, G., AND STETINA, R. (1997) Mutation Research 375, 183-193.
9. Comet assay references, procedures and information can be found at two websites:
A) Comet Assay Interest Group (NIH) http://cometassay.com/
B) Comet Assay Forum (Industrial Toxicology Research Center, Lucknow, India) http://www.comet.itrcindia.org/ - Dhawan, A.,
Bajpayee, M., Pandy A. K., and Parmar, D., The SCGE/Comet Assay Protocol, ITRC.
10. NADIN, S. B., VARGAS-ROIG, L. M., AND CIOCCA D. R. (2001) The Journal of Histochemistry & Cytochemistry 49, 1183-1186.
11. KOPPEN, G. (1997) Comet Newsletter, Kinetic Imaging Ltd., 6, 2-3.

©2006 USB Corporation–All rights reserved.

USB and the logo are registered trademarks of USB Corporation.
Triton is a registered trademark of Union Carbide Chemicals and Plastics Company.
Uracil DNA Glycosylase - A license may be required from Invitrogen Corporation to practice the process claimed in U.S. Patent No. 5,035,996.
SYBR is a registered trademark of Molecular Probes, Inc.

USB Corporation
26111 Miles Road, Cleveland, Ohio 44128
www.usbweb.com F u e l i n g I n n o v a t i o n™
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