4. Results

co-workers, the first to describe an alkaline version of the assay, specify that electrophoresis is carried out for 20 mins at 25 V, at 300 mA, and a depth of liquid of 0.25 cm [2]. Since the tank dimensions are not known, the local electric potential is undefined. In a follow-up review paper, the electrophoresis is at 12 V and 100 mA, with the circulation of the electrophoresis solution at 100 ml/min of a total volume of 1 liter [10]; again, this implies an undefined electric potential. In a later publication [11], the same group describes electrophoresis at 12 V (0.4 V/cm), 250 mA, and a

In guidelines from 2000 [12], R.T. Tice (who was also co-author of the 1988 protocol with N. P. Singh [2]) states that "… due to the large variability in the size of commercially available electrophoresis units, it is more accurate and useful to present the voltage in V/cm". Concerning voltage, 0.7–1.0 V/cm and a duration of 5–40 mins are specified, and circulation (whereas current, liquid volume, and depth are not mentioned in these guidelines) [12]. In the comet assay later described by Peggy Olive [3, 13], electrophoresis was run at 1 V/cm for 20 min (nothing said about volume and current). In a follow-up protocol by Peggy Olive in 2006 [14], electrophoresis is conducted at 0.6 V/cm for 25 min; "The current should be about 40 mA if using 20 V. The distance in centimeters is measured between the negative and positive electrophoresis in the electrophoresis chamber." There is a problem associated

Among the early comet assay protocols, the most cited ones (by March 2018) are [2] (6522 citations) and [12] (2906 citations); citations for Östling & Johansson [1] are not available. Judging from these bibliographic data, and since the electrophoresis conditions in [2] are undefined with respect to electric potential but recommending a specific current (300 mA), it seems that much of the confusion in the literature stems from the uncritical reference to [2]. A more frequent use of the guidelines [12] might have avoided some of the unex-

The current plays no direct role in electrophoretic mobility, but may indirectly affect the local distribution of the electric potential in a tank. The organisation for economic co-operation and development (OECD) In vivo comet assay Test Guidelines protocol 489 [15] from 2014, correctly states that the "… level of DNA migration is linearly associated with the duration of electrophoresis, and also with the electric potential (V/cm)." However, there is still the recommendation of a "starting current of 300 mA (—), the depth should be adjusted to achieve these conditions, and the current at the start and the end of the electrophoresis should be recorded". It is worth noticing, however, that attempts to control the current often lead to very shallow liquid levels. In [16] the level of the buffer is described as "… about 2–3 mm above the agarose on the slide"; in [2] the depth was 0.25 cm. It is often underlined in the protocols that the electrophoresis tank must be in the level, so that the depth above the gels is equal over the platform surface. However, keeping a large tank level in each run, within an error of, for example, 1 mm is hardly achievable. The implication is that the resistance of the liquid on the platform will vary relatively much, leading to potentially large differences in local electric potential. Specifying a much higher depth, for example, 6–10 mm solves this problem, although a larger current would put demands on the power supply. We will get back to this problem in

duration of 20 mins.

66 Electrophoresis - Life Sciences Practical Applications

with this approach, which we will discuss later.

the recommendations at the end of this text.

pected comet assay results published during the last 20–30 years.
