Comet Assay Kit - Single Cell Gel Electrophoresis (SCGE)
$285.00
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Cat. No.: SCGE-50 (for 50T)
Description
The comet assay, also known as single-cell gel electrophoresis (SCGE), is a commonly used technique for measuring DNA damage in individual cells. When cells experience DNA strand breaks caused by various endogenous and exogenous DNA-damaging factors, their supercoiled DNA structure is disrupted. Under an electrophoretic field, the damaged DNA migrates toward the positive electrode, forming a "comet" image, while the undamaged DNA remains spherical. Under a microscope, this results in a "tail" resembling a comet. The severity of DNA damage is indicated by the length of the comet tail, with longer tails representing more severe damage. Various parameters can also be measured using image analysis software.
Components
50T
- 10X Neutralization Solution
- 10000X DNA Dye
- 20X Electrophoresis Buffer
- 10X Lysis Solution
- 10X EDTA
Storage
This kit is valid for one year from the date of purchase.
Notes
- The lysis solution may become cloudy at room temperature and should be stored at 4°C or temporarily at -20°C.
- Different cell types and treatment methods can affect lysis and electrophoresis times.
- The number of cells seeded should not be excessive.
Protocol
Spread a thin layer of normal melting point 0.5% agarose on a microscope slide and allow it to solidify.
Mix the cell suspension with 2% low melting point agarose at a 1:1 ratio, then spread it on the prepared slide and allow it to solidify.
After the agarose containing cells has solidified, immerse it in lysis buffer at pH 10.0 for 1.5 hours, then wash with pre-chilled distilled water three times, each wash lasting 20 minutes.
1X Lysis Buffer Preparation:Dissolve in approximately 60 ml of distilled water, adjust the pH to 10.0 with NaOH, and make up to 100 ml. Store at 4°C until use.- NaCl: 14.61 g
- 10X EDTA: 10 ml
- 10X Lysis Buffer: 10 ml
- DMSO: 1 ml
- Incubate in pre-chilled electrophoresis buffer for 45 minutes, then perform electrophoresis at 25V for 25 minutes.
- 1X Electrophoresis Buffer Preparation: Add 850 ml of distilled water to 50 ml of 20X electrophoresis buffer, dissolve, adjust the pH to 12.8 with NaOH, and make up to 1000 ml. Store at 4°C until use.
- After electrophoresis, immerse the slide in neutralization buffer for 1 hour.
Neutralization Buffer Preparation: Add 400 ml of distilled water to 50 ml of 10X neutralization buffer, dissolve, adjust the pH to 7.5 with HCl, and make up to 500 ml. Store at 4°C until use.
Stain with DNA dye for 15 minutes, then wash with PBS three times, each wash lasting 5 minutes.
Observe and photograph under a fluorescence microscope.
Only for research and not intended for treatment of humans or animals
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