Comet Assay Systems: "Comet" Single Cell Gel Electrophoresis Systems

The COMET single cell gel electrophoresis systems are designed specifically for single cell gel electrophoresis (SCGE). Comet Assays are used to detect and quantify DNA damage and repair within individual cells in genetic toxicology and carcinogenesis studies.

comet gel electrophoresis unit Features/Benefits:
• Available in 2 formats - to accommodate 20, or 40 standard microscope slides respectively
• Opaque ebony acrylic tank and lid - provide ideal protection for techniques requiring light-sensitive stains or samples
• Cooled central platform - provides a convenient surface for slide preparation in addition to maintaining slide temperature during pre-incubation and electrophoresis (see below)
• Cooling ports - can be connected to the JULABO chiller unit: to prevent overheating during SCGE/Comet assays, typically performed at high current settings over 300mA; and to inhibit DNA repair enzyme activity by maintaining the slide temperature at 4°C, either during preparation and mounting or preincubation and electrophoresis
• Buffer recirculation ports - can be connected to a peristaltic pump to maintain buffer circulation, preventing ion gradient formation
• Colour-coded handles - corresponding to the anode and cathode - serve as a visual aid to ensure that the slides are positioned in the correct polar orientation for the assay

Background - The Comet Assay/SCGE was pioneered by Östling and Johansson (1) in 1984 as a neutral pH assay to quantify double-stranded DNA breakages (DSBs) in single cells exposed to γ irradiation. The system was then modified in 1988 to a more versatile and sensitive alkaline method to measure both single- (SSBs) and double-stranded DNA breakages. Now, by introducing subtle changes to the assay conditions (pH/temperature), the Comet Assay can be tailored to analyse specific DNA lesions and repair processes.

Overview - The Comet Assay is based on the principle that strand breakage of supercoiled duplex DNA reduces the size of the large genomic DNA molecule from which these strands are separated or stretched out by electrophoresis. The high pH of the alkaline lysis step causes denaturation, unwinding of the duplex DNA and the release of alkali labile sites as SSBs. These then become “comets” as the broken ends of the negatively charged DNA molecule migrate towards the anode during electrophoresis.

Method - Cells, previously exposed to a genotoxic insult, are suspended in low melting point agarose (LMP) and embedded within a thin agarose gel on a microscope slide. All cellular proteins are then removed by lysis in detergent solution, when the DNA is allowed to unwind in the neutal/alkaline pH conditions of the detergent. The DNA is electrophoresed and then stained with a DNA-specific fluorescent dye such as acridine orange, ethidium bromide, propidium iodide or 4',6'-diamidino-2- phenylindole (DAPI).

Result - Upon staining cellular DNA is measured for fluorescence, usually with a microscopy imaging system. The resulting image resembles a “comet”, with the cellular DNA segregating into a “head” and “tail”. The head is composed of largely intact genomic DNA, while the tail comprises damaged (SSBs, DSBs) or fragmented DNA, with the fluorescence intensity and length of the tail being directly proportional to the extent of the DNA damage.

 


Model:
COMET-20
COMET-40
Unit Dimensions (W x L x H) 31 x 32 x 6.5cm 31 x 47.5 x 6.5cm
Active Tank Dimensions (W x L x H) 27.5 x 13 x 3.5cm 27.5 x 37 x 3.5cm
Recommended Buffer Volume (ml) 600ml 800ml
Buffer Recirculation Ports 2 2
Slide Capacity (25 x 75mm; W x L) 20 40
Recommended Running Conditions 5V/cm (300mA) 5V/cm (300mA)
Power Output Connectors (diameter) Shrouded, 4mm Shrouded, 4mm
Recommended Power Supplies Consort EV261 Consort EV261


Ordering Information
COMET 20 Complete system for 20 Slides $580.00
COMET 40 Complete system for 80 Slides $705.00


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