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Materials and Protocols

Comet assay does not require any special apparatus or chemicals. It can usually be performed on materials found in most laboratories. Based on our experience and that of others we recommend the use of the following for reliable and reproducible comet assay results.

Comet Assay Slides: Regular unpolished glass microscope slides work well for routine Comet assay use. For Comet assays that require extensive washes like comet-FISH, glass microscope slide from Erie Scientific (Cat no. XES 370 and XES 368). is recommended. These slides are frosted in the periphery and the gels cast on them can withstand repeated washes. TrevigenŽ slides are very convenient for treating cells with DNA repair enzymes. Moreover Trevigen slides does not require pre-coating with normal melting point agarose.

Electrophoresis Tank: Horizontal electrophoresis tank with passive re-circulation from Fisher Scientific (FB-SBR-2025) or Owl Scientific is widely used. Electrophoresis is routinely performed at 25 V and 300 mA using this electrophoresis tank. Other tanks can also be used, but voltage and current conditions need adjustment.

Miscellaneous: 1. Aluminium slide trays ( Shandon Lipshaw) 2. Plastic slide trays (Shandon Lipshaw) 3. Slide Marker: SecurlineŽ

Different versions of Comet Assay

1. Alkaline Comet Assay

2. Neutral Comet Assay

3. Comet assay at pH 12.1

4. Comet assay with DNA repair enzymes

5. Acellular Comet Assay

6. Comet-FISH Assay

7. Bromodeoxyuridine Labeling to Detect Replicating DNA

8. Detection of incomplete DNA excision repair sites

9. Halo assay or non-electrophoretic assay

 

ALKALINE COMET ASSAY (pH > 13)

1. Ray Tice and Marie Vasquez 

2. TrevigenŽ Kits:  A, B, C, D and E

 


ALKALINE COMET ASSAY (pH ~ 12.1)

Similar to Alkaline Comet assay except requires pH of unwinding and electrophoresis buffer to be adjusted to 12.1 with 1 M HCl. It is believed that at pH 12.1, the alkali labile sites are not expressed.


NEUTRAL COMET ASSAY (pH 7-10)

Olive and Banath, 1993 or appropriate modifications


COMET ASSAY WITH DNA REPAIR ENZYMES


MORE COMET ASSAY PROTOCOLS (INDIA COMET ASSAY FORUM)