Help & FAQs: DNA Damage and Repair Kits

CometAssay Single Cell Gel Electrophoresis Assays

  • Can 2-Well CometSlides™ be used instead of the 3-Well FLARE™ Slide?
  • The well size for the CometSlide™ 2- well and the FLARE™ Slide 3-well are identical. The slides are interchangeable.
  • Can the Alkaline CometAssay® Control Cells be used for the Neutral CometAssay?
  • The Alkaline CometAssay Control Cells were qualified for the Alkaline CometAssay while the Neutral CometAssay Control Cells were qualified for the Neutral CometAssay.
  • Can the CometChip® be stored before staining?
  • The neutralized gel can be stored in 20 mM Tris pH 7.4 buffer at 4 °C for a few days.
  • Do you recommend use of the CometAssay® with whole blood?
  • We do not recommend using whole blood with the CometAssay because sample preparation is critical, and hemoglobin could damage DNA. Additionally, it is important to note that vertebrate red blood cells (major blood component) do not have nuclei (except for birds) and therefore are not suitable for use with the CometAssay.
  • How do I avoid loss of agarose disks from the CometSlides™?
  • FLARE™ and CometSlides™ are specially treated to enhance agarose binding during alkaline treatment.Recommendations:Special attention needs to be paid to spread the agarose/cell mixture evenly over the entire slide well. Be careful to avoid getting agarose on the colored portion of the slides.Always place the slides into solutions; never pour or decant buffer over comet slides.Minimize time slides spend in alkaline buffer. Follow recommended incubation times.Rinse and suspend cells in PBS to remove medium before combining with LMAgarose.Do not increase ratio of cells to molten agarose by more than 1 to 10.Place slides at 4 °C for 15 min to assure complete gelling of the agarose before lysis.
  • How long can FLARE™ Slides or CometSlides™ be stored after electrophoresis?
  • Prior to staining, slides immersed in 70% ethanol for 5 minutes and then dried can be stored at room temperature for 1 year prior to staining. Permanent records can be created after staining for visualization by standard light microscopy using the CometAssay® Silver Staining Kit (Catalog # 4251-050-K).
  • How should the CometAssay® Control Cells be stored?
  • The CometAssay Control Cells should be stored in liquid nitrogen. To avoid the accumulation of damage due to freeze thaw cycles, the CometAssay Control Cells should be thawed and frozen in working aliquots. Refer to the CometAssay Control Cells literature for specific instructions.
  • Is it necessary to use a coverslip with CometSlides™?
  • FLARE™ and CometSlides™ were designed to be used without coverslips because removal of the coverslip could cause detachment of the agarose and cause damage to the samples.
  • Is it possible to combine the Anti-fade and SYBR® GOLD/SYBR® Green?
  • The solutions can only be mixed prior to application. Precipitation occurs upon storage of Anti-fade and SYBR mixture. 
  • Is it possible to re-use a FLARE™ slide or CometSlide™ if not all wells are used?
  • FLARE™ and CometSlides™ contain a special coating that promotes agarose binding, which can be compromised if washed for re-use.
  • Is it possible to reuse a CometChip® if not all wells are used?
  • The CometChip cannot be reused. After treatment and before lysis, the CometChip is covered with LMAgarose, which will fill the micropores in any unused wells.
  • Is it possible to treat cells in the CometChip® for longer than the recommended time?
  • The protocol will be reflective of what we have validated in-house for best results. If longer treatment times are required, the treatment step should be carried out in a tissue culture plate, after which time the cells can be transferred to the CometChip.
  • Is there a convenient step to stop the CometAssay® and resume the next day?
  • The lysis protocol can be performed at 4 °C for 30-60 minutes or overnight. Otherwise, except for staining, the entire CometAssay® protocol must be completed until slides containing samples are completely dry. Slides may be stored at room temperature, with desiccant, prior to scoring at this stage.
  • The silver staining protocol for the CometAssay® requires 100 µL per well staining volume for two-well slides. What is the staining volume for twenty-well slides?
  • Silver staining volumes for the CometAssay can be reduced to 50 µL per well when using a twenty-well slide.
  • What are the dimensions of the CometSlides™?
  • The 2- and 3-well CometSlides have the same dimensions as standard microscope slides, 75 mm x 25 mm x 1 mm. The 20-well CometSlide™ is 75 mm x 50 mm x 1 mm. The 96-well CometSlide is 122 mm x 78 mm x 1 mm.
  • What cell line is used to generate the CometAssay® Control Cells?
  • CometAssay Control Cells were prepared using an immortalized human lymphocyte line.
  • What CometAssay® Electrophoresis Starter Kit is appropriate for use in Korea?
  • The CometAssay® Electrophoresis Starter Kit plus EU Power Supply (Catalog # 4250-050-ESK-PS2) is compatible with Korean outlets and voltage. Ensure voltage on the back of the power supply is set to 230 V before use.
  • What DNA damaging agent was used to create the CometAssay® Control Cells?
  • A healthy control cell population (C0) is treated with a DNA damaging agent to increase the amount of damage in populations C1, C2 and C3, respectively. Etoposide is the damaging agent used for Alkaline Control Cells (CC0-CC3) and Bleomycin is the damaging agent used for Neutral Control Cells (NC0-NC3).
  • What image analysis tools are available for analysis of data generated by the FLARE™ Assay, CometAssay®, and CometChip®?
  • We offer a software (Catalog # 4260-000-CS) designed to improve, standardize, and make comet analysis easier. The software will automatically locate and score comets from digital images to characterize and quantify the degree of DNA damage revealed by the comet assay. It is compatible with FLARE Assay, CometAssay, and CometChip Kits. The Comet Analysis Software can rapidly evaluate large numbers of cells and generate summary statistics based on the corresponding numeric results.
  • What is the difference between the CometChip® and the 96-well CometSlide™?
  • The 96-well CometSlide is employed in the traditional comet assay and requires each cell population to be treated and resuspended in molten LMAgarose individually before pipetting onto the CometSlide.In contrast, the CometChip uses gravity to capture cells in agarose pre-stamped with micropores for treatment directly within the slide, allowing for easier high throughput studies. After inserting the CometChip into the 96-well macroformer, cells are added to each well. Each macrowell of the CometChip contains ~400 non-overlapping micropores that capture a single cell for use in the comet assay. The cells can be treated, lysed, and electrophoresed directly on the CometChip.
  • What is the importance of pH for the alkaline electrophoresis buffer in the Comet Assay?
  • The pH of the electrophoresis buffer will determine the types of DNA damage that can be analyzed. At pH 12.1, initial breaks are analyzed, while at pH 12.5 and pH 13 alkaline labile adducts are converted to breaks. At pH 12.5, basic lesions are converted to single strand breaks and at pH 13, additional labile sites are converted to single and double strand breaks. Maximum damage caused by an agent is visualized at pH 13 in FLARE™ Assay, CometAssay®, and CometChip® Kits.
  • What is the optimal filter set for SYBR® GOLD?
  • SYBR® Gold’s maximum excitation/emission is 496 nm/522 nm. A fluorescein filter should be adequate.
  • What is the purpose of the CometAssay® Control Cells?
  • The CometAssay Control Cells are designed to help standardize and compare alkaline and neutral electrophoresis methods between individual users and laboratories. The control cells contain known levels of damage.
  • What is the size of a single pore in the CometChip®?
  • The CometChip micropores are 30 µm in diameter and 50 µm deep.
  • What staining alternatives to SYBR® Gold are suitable for the CometAssay®?
  • SYBR Gold nucleic acid gel stain (10,000X concentrate in DMSO) is recommended. SYBR Green can also be used.Less sensitive alternatives include the following:Acridine Orange (50 µM)- single strand breaks appear red and double strand breaks appear yellow–green.Ethidium Bromide (20 µg/ml) –binds double strand breaks more efficiently than single strand breaks.DAPI (1 µg/ml) - binds to major groove of DNA and fluorescence is dependent on double-stranded structure.Propidium Iodine (2.5 to 20 µg/ml) – binds to DNA by intercalating between bases.Hoechst 33258 (0.5 µg/ml) – binds to minor groove of DNA.YoYo-1 – binds to DNA by intercalating between bases.  R&D Systems has not tested YoYo-1 in house, so there is no recommended concentration.
  • Why are comet tails in positive control cells not present or smaller than expected?
  • Failure to lyse, denature in alkali, or properly perform electrophoresis may generate poor results.  To improve results:Verify lysis of cells. Lysis Solution should be stored long term at room temperature and chilled to 4 °C prior to use. Lysis Solution stored long term at 4 °C will precipitate. Try overnight lysis at 4 °C.Perform alkali denaturation for 20 minutes at room temperature or for at least 1 hour at 4 °C. Prepare Alkaline Solution fresh from sodium hydroxide pellets.Electrophoresis conditions are also critical for optimal performance. Conventional slab gel electrophoresis chambers are not designed to eliminate known causes of comet assay variability (alkaline pH, buffer height, temperature, slide orientation). For optimal results, use a CometAssay Electrophoresis system (Catalog # 4250-050-ES). For insufficient electrophoresis time, increase time up to 1 hour at 4 °C for alkaline electrophoresis.
  • Why are there comet tails in the healthy CometAssay® Control Cell population?
  • The CometAssay Control Cells were prepared from an asynchronous cell population with cells at all stages of the cell cycle. Some percentage of the population will be undergoing DNA replication and may have strand breaks at replication forks.Improper storage may also lead to DNA damage. Do not store CometAssay Control Cells at -80°C. Store in aliquots in liquid nitrogen.
  • Why don't I see a visible cell pellet after centrifugation of the CometAssay® Control Cells?
  • A pellet amy not be visible after centrifugation due to the low number of cells.
  • Why is the cell culture medium leaking out from the wells of the CometChip® macroformer?
  • There are a few things that may lead to leaking in the macroformer:Treatment time is too long. The macroformer is designed for ~30 minute loading time followed by ~1 hour treatment time. Longer treatment times may result in leaking. If longer treatments are required, the treatment step should be carried out in a tissue culture plate, after which time the cells can be transferred to the CometChip.The slide was not properly placed into the macroformer. If the slide is not positioned properly on the black bottom, the macroformer will not snap into place to properly make a seal. Ensure that the macroformer is evenly spaced above the black base. Always carry the unit by the black base. If held by the white macroformer, the magnets will separate and cause leakage.The CometChip is expired. The CometChip has an 8-week shelf life. Expired CometChips begin to dry out and will not form a proper seal with the macroformer.
  • Why is it necessary to rinse the CometAssay® Control Cells in PBS before adding to LMAgarose?
  • The CometAssay Control Cells must be rinsed in PBS to eliminate media and storage factors that reduce adherence of LMAgarose to the slide. PBS without magnesium should be used, as magnesium stimulates nucleases.
  • Will a ZEISS Axioplan/Axiovert microscope work for viewing CometSlides™?
  • Yes, the ZEISS Axioplan/Axiovert microscope will work for viewing a FLARE™ or CometSlide™.
  • Will any commercial horizontal gel electrophoresis device work for the Comet Assay?
  • Electrophoresis conditions described for FLARE™ Assay, CometAssay® and CometChip® Kits were optimized for the CometAssay® Electrophoresis System (Catalog # 4250-050-ES). Conventional slab gel electrophoresis chambers are not designed to eliminate known causes of comet assay variability (alkaline pH, buffer height, temperature, slide orientation). They can be used, but they require optimization by the user to achieve consistent results.

DNA Damage and Repair Assays

FLARE Assay Kits