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In the alkaline comet assay, cells are embedded in agarose, lysed, and then subjected to further processing including electrophoresis at high pH (>13). We observed very large variations of mean comet tail lengths of cell samples from the same population when spread on a glass or plastic substrate and subjected to electrophoresis. These variations might be cancelled out if comets are scored randomly over a large surface, or if all the comets are scored. The mean tail length may then be representative of the population, although its standard error is large. However, the scoring process often involves selection of 50 – 100 comets in areas selected in an unsystematic way from a large gel on a glass slide. When using our 96-sample minigel format (1), neighbouring sample variations are easily detected. We have used this system to study the cause of the comet assay variations during electrophoresis and we have defined experimental conditions which reduce the variations to a minimum. We studied the importance of various physical parameters during electrophoresis: (i) voltage; (ii) duration of electrophoresis; (iii) electric current; (iv) temperature; and (v) agarose concentration. We observed that the voltage (V/cm) varied substantially during electrophoresis, even within a few millimetres of distance between gel samples. Not unexpectedly, both the potential ( V/cm) and the time were linearly related to the mean comet tail, whereas the current was not. By measuring the local voltage with microelectrodes a few millimetres apart, we observed substantial local variations in V/cm, and they increased with time. This explains the large variations in neighbouring sample comet tails of 25% or more. By introducing simple technology (circulation of the solution during electrophoresis, and temperature control), these variations in mean comet tail were largely abolished, as were the V/cm variations. Circulation was shown to be particularly important and optimal conditions were established. Some further improvements were obtained by standardising the procedure for making agarose gels. In these experiments, we considered the comets as “dead” objects obeying the laws of physics and chemistry, since lysed cells are devoid of most proteins at pH>13 and enzymatic processes are not likely to take place. In total, the variations of mean comet tails in neighbouring samples were reduced to a level at which “true” biological variations of the cell population will dominate when subjected to analysis in the comet assay.