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A guide on scoring a comet slide

If you are new to the comet assay and unsure how to go about scoring your comet slide, hopefully this article will help.

Lights, camera, action!
First, check that you are illuminating the comets correctly.  For example, the correct coloured lamp with the appropriate DNA stain. Note that bright lights may cause the DNA stains to degrade (lose their florescence) and so it is important you don’t leave the comet slide under the microscope for longer than necessary. 
If you are using Comet Assay IV with the live-video imaging feature, check your camera is connected and the software is ready to be used. 

Check you are using the correct magnification.
Typically, an objective x10 or x20 will provide you with nicely sized comets.   However, this could vary depending on your cell type. 
If you recently purchased Comet Assay IV and have specific questions about this, please ask us. 

Focus!
To gain the most accurate results, it is important you are viewing optimally focussed comets.  Depending on the thickness of your gel, it may be necessary to re-focus your image between different comets.  

Don’t score cells at the gel edge.
Towards the edges of your gel, your comets may appear distorted; this could be due to the drying effect on the gel on the glass slide.  To avoid measuring these comets, adjust the microscope stage so you are looking at a section of gel away from the edge.  Then, plan your journey across the microscope slide avoiding any edges.  The same advice should be followed if you have any other imperfections in the gel, such as cracks, damage or extra think gel blobs.  Scoring cells which are not representative of your data set may skew your results.

How to move the microscope stage
It is best to move around your gel in a logical and methodical fashion.  This helps to avoid scoring a single comet multiple times. 
Position your microscope slide and stage so you can view your comets.  Most people like to start in the ‘top left’ of the gel (avoiding the absolute edge of the gel, as mentioned above).  Then journey across the slide to the ‘top right’, scoring comets as you go.  Now, adjust the stage so you are viewing comets slightly below the ones you just scored, staying on the right side of the gel.  Journey back across the stage to the left side, again, scoring any appropriate comets.  Then, continue moving back and forth across the slide, getting further and further towards the bottom of the gel.  Continue until you have scored the required amount of comets.  Using this technique, you can be sure each comet is scored only once.

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We regularly attend comet assay workshops and contribute to the debate towards regulatory acceptance of the assay.

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