Are there other dyes beside SYBR Gold that can be used in the Comet Assay?

Yes, other dyes that can be substituted for SYBR Gold in staining solution for the Comet Assay. The important parameters to consider in choosing a DNA stain for the comet assay are similar fluorescence and decay rates for single-and double-strand DNA. See Appendix C in the product insert for a table of DNA stains parameters. R&D Systems selected SYBR Gold dye due to the high background to noise ratio. It is the responsibility of the end-users to optimize the use of an alternative dye chosen for the Comet Assay.

Do control cells, Catalog # 4256-010-CC, just need to be put on a slide then stained? Or do they need to be ran through the entire Catalog # 4250-050-K protocol to see comets?

The control cells, Catalog # 4256-010-CC, need to be run through the entire protocol for Catalog # 4250-050-K in order to see comets.

How does one correct the problem of seeing cells in multiple planes of view when using the CometAssay, Catalog # 4250-050-K?

The main fix for this issue is to ensure that the agarose is completely dry. Depending on the lab environment, it may take longer than the suggested 10-15 minutes indicated in the protocol.

What are the slide dimensions for the CometSlides (Catalog # 4250-200-03)?

The CometSlides (Catalog # 4250-200-03) are 75x25x1 mm with a 1.4 cm well diameter.

What can cause observation of artefacts during imaging of Comet data?

For optimal imaging, all cells should be in the same plane and this is achieved by completely drying the agarose. Artefacts may be introduced if the source cells used for analyis has any debris. The debris should be allowed to settle before pipetting cells. The LMagarose can also be the cause of artefacts. To prevent debris from accumulating in the agar, melt it at 80-90 °C so it is runny, spin it at low speed and aliquot into sterile tubes in a hood. Use each aliquot for a single assay.

CometAssay Single Cell Gel Electrophoresis Assay Best Seller

Name: CometAssay Single Cell Gel Electrophoresis Assay
Brand: R&D Systems
SKU: 4250-050-K

R&D Systems, part of Bio-Techne | Catalog # 4250-050-K

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Product Summary
Kit Contents
Preparation & Storage
Product Documents

Key Product Details

Features

Includes twenty-five specially treated CometSlides (2 Well) and sufficient reagents for 50 test samples.

Key Benefits

Specially treated slides to promote agarose adherence
Ready to use low melt agarose (LMAgarose) in convenient size
Lysis Solution suitable for either alkaline or neutral comet assay
Optimized electrophoresis conditions

Product Summary for CometAssay Single Cell Gel Electrophoresis Assay

Product Specifications

Applicable for the analysis of either single- or double-strand DNA breaks
Study cellular responses to DNA damage
Screen for inhibitors of DNA repair
Test chemicals for toxicity
Screen for environmental mutagens

What is the CometAssay?

The Comet assay, or single cell gel electrophoresis assay, provides a simple and effective method for evaluating DNA damage in cells. The principle of the assay is based upon the ability of denatured, cleaved DNA fragments to migrate out of the nucleoid under the influence of an electric field, whereas undamaged DNA migrates slower and remains within the confines of the nucleoid when a current is applied. Evaluation of the DNA “comet” tail shape and migration pattern allows for assessment of DNA damage. The general Comet assay protocol includes first immobilizing cells in a bed of low melting point agarose, gently lysing the cells, treating cells with neutral or alkali solutions to unwind and denature the DNA as well as hydrolyze sites of damage. DNA is then subjected to electrophoresis and stained with a fluorescent DNA intercalating dye.

Kit Contents for CometAssay Single Cell Gel Electrophoresis Assay

CometAssay Lysis Solution
CometAssay LMAgarose
CometSlides (2 Well, 25 Slides)
200 mM EDTA, pH 10

Formulation, Preparation, and Storage

Shipping

The product is shipped ambient or with polar packs. Upon receipt, store it immediately at the temperature recommended on the product label.

Storage

Store products immediately at the temperature recommended on the product labels.

CometAssay Single Cell Gel Electrophoresis Assay Best Seller

R&D Systems, part of Bio-Techne | Catalog # 4250-050-K

Key Product Details

Features

Key Benefits

Product Summary for CometAssay Single Cell Gel Electrophoresis Assay

Kit Contents for CometAssay Single Cell Gel Electrophoresis Assay

Formulation, Preparation, and Storage

Shipping

Storage

Product Documents for CometAssay Single Cell Gel Electrophoresis Assay

Product Datasheets

COA

Certificate of Analysis

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