Troubleshooting guide
Sample Preparation
- Cells suspended within the LMAgarose did not remain attached to the glass slide.
Possible Causes
Electrophoresis solution too hot.
Cells were not washed to remove growth medium before combining with LMAgarose.Action
Control temperature by recirculating the electrophoresis solution or performing the assay at less than 20°C.
LMAgarose percentage was too low.Action
The pH of medium and carry over serum proteins, etc., can reduce the adherence of the agarose. Resuspend cells in 1X PBS prior to mixing with the LMAgarose.
The LMAgarose was not completely set before the samples were processed.Action
Adjust the ratio, but do not increase ratio of cells to molten agarose by more than 1 to 10.
LMAgarose unevenly set on the slide.Action
As the LMAgarose sets, it appears to shrink; ensure a 0.5 mm dried ring due to agarose retraction is seen at the edge of the sample.
LMAgarose was not completely melted before use.Action
Spread the agarose with the side of a pipette tip to ensure uniformity of the LMAgarose and better adherence to the glass slide.
Slides are not adequately NMAgarose-precoated or the humidity is too high.Action
Completely solubilise the agarose before transferring to a 37°C water bath.
Slides are not handled gently during lysis and electrophoresis.Action
Ensure pre-coating of glass slide with NMAgarose has covered slide uniformly and dried completely.
Action
Take care during comet assay procedure. Do not pour solutions directly over slides or drop slides (i.e. into coplin jars or containers). Instead, remove slides and then slowly immerse slides in pre-filled container or jar.
- LMAgarose contains bubbles
Cause
Air introduced to sample during preparation and/or LMAgarose too cool during handling
Action
Hold LMAgarose at 37°C and immediately add to slide.
Minimize pipetting and mixing of sample with LMAgarose.
Warm slide to 37°C to ensure even coating of well.
- In the negative control, the majority have large comet tails.
Cause
Unwanted damage to cells has occurred during sample preparation.
Action
Check morphology of cells to ensure healthy appearance before sample preparation.
Handle cells gently to avoid physical damage.
Ensure electrophoresis buffer solution remains below 20°C.
Keep cells on ice as much as possible and prepare cell samples immediately before combining with molten LMAgarose.
If an enzyme disaggregation method is used, try reducing exposure time or enzyme concentration.
- In the negative control, the majority have small to medium comet tails.
Cause
The DNA has been damaged: Endogenous oxidative damage or endonuclease activity after sample preparation is damaging DNA
Action
Ensure the lysis solution was chilled before use.
If a PBS solution is used, ensure it is calcium and magnesium free.
Work under dimmed light conditions or under yellow light.
Add DMSO to any cell sample that may contain heme groups.
- Unexpected and/or variety of tail shape
Cause
LMAgarose used was too hot.
Action
Cool LMAgarose to 37°C before adding cells.
- In positive control (e.g. extreme hydrogen peroxide treatment) there is no evidence of comet tail
Cause
The cellular DNA has not been damaged, it is most likely that the sample was not processed correctly
Action
Ensure a fresh hydrogen peroxide solution is used to induce damage. Hydrogen peroxide solution should be stored at 4°C, tightly capped once opened.
Increase treatment time.
Increase hydrogen peroxide concentration.
Ensure each step in protocol was performed correctly. Failure to lyse or to properly perform electrophoresis may generate poor results.
- In the positive control (e.g. extreme hydrogen peroxide treatment) comet tails are present but not significant.
Possible Causes
The degraded DNA has not migrated far enough from the comet head due to an insufficient electrophoresis time.
Insufficient denaturation in alkaline solution.Action
Increase time of electrophoresis (up to 20 minutes for TBE and up to 1 hour for alkaline electrophoresis). Note: the electrophoresis time can be increased when running at a cold temperature (4°C).
Action
Increase time in alkaline solution up to 1 hour.
- Results are not reproducible.
Possible Causes
LMAgarose concentration is variable.
Errors occurring in sample preparation.Action
Ensure that LMAgarose is fully dissolved and the concentration has not been altered (e.g., by evaporation).
Depth of electrophoresis buffer is variable across the bed.Action
Ensure the method is followed accurately for each sample and replicate.
LMAgarose is formed without using a coverslip.Action
Use a spirit (bubble) level device to ensure that the electrophoresis chamber is level, a covering of each slide 1mm deep is sufficient.
The solutions are ‘old’.Note
If a coverslip is not used to flatten out the LMAgarose, it can solidify in a blob-like manner. This means that the comets in the centre of the thick LMAgarose blob may appear different to those located at the thinner edges.
Action
Adjust method to use a coverslip.
Errors occurring during methodAction
Typically, the solutions used should be no older than 1 month old.
Action
Ensure the method is as reproducible as possible. Unequal unwinding times, pH or electrophoresis time can alter the results dramatically.
Image Acquisition
- The image is not obtainable
Possible Cause
There are many factors which need to be correct to ensure the comets can be analysed, including but not limited to:
1) A correctly optimised microscope
2) Correct illumination and filters
3) A suitable adapter to connect the camera to the microscope
4) Microscope camera port not set to send image to the cameraAction
It is essential to maintain a regular service schedule for your microscope and lamp. Read our comet assay microscopy guide (see menu on right) for more information.
- No, or poorly visible, comets are observed
Cause
Insufficient staining
Action
Increase staining time or concentration of DNA stain. As far as Comet Assay IV is concerned, any stain is acceptable. Although the majority of our customers use one of the following fluorochromes for their comet slides: Ethidium bromide, Propidium iodide, SYBR Green or SYBR Gold. Regardless of the stain used, there are some important factors to take into account. Read our comet assay microscopy guide (see menu on right) for more information.
Poor Image Quality
- Blurry comet.
Possible causes
Incorrectly focussed microscope.
An unclean lens system in the microscope.Action
Re-take image (or refocus microscope live feed) with correct focus in place.
The exciter filter and barrier filter are incorrectly combined.Action
The optical elements of a microscope should be completely free of dust, dirt, oil, solvents, and any other contaminants.
The aperture iris diaphragm or field iris diaphragm opening is not opened correctly.Action
Follow the filter combinations for the fluorochrome.
Action
Open the aperture iris diaphragm completely, and open the field iris diaphragm until its image circumscribes the field of view.
- The comet is too dark.
Possible causes
Poor camera setting.
Insufficient DNA staining.Action
Increase the shutter value to brighten the image.
The microscope lamp needs replacing.Action
Apply a higher concentration or longer staining time.
Action
Arrange a regular service for your microscope and lamp.
- The background is too bright.
Possible cause
There are several reasons why the background may be bright, including poor camera setting or sample preparation.
Action
Reduce the shutter value and increase the gain to darken the image. Stray infrared light entering the microscope may cause a bright background or the fluorescence of debris introduced during the sample preparation.
Abbreviations:
LMAgarose = Low melting point agarose
NMAgarose = Normal melting point agarose