I am performing a comet assay and as a positive control using 50 micromolar H2O2 treated cells. Though DNA damage is seen, the direction of the comet tails is somewhat diagonal and not straight towards positive electrode.
I am performing electrophoresis at ~50mA current for ~ 20 mins at an 1V/cm. The distance between 2 electrodes is about 14 cm.
What could the reason for the directionality be?
Dear Paromita, it's perhaps because your slides slightly moved during electrophoresis. You can avoid this problem by adding slides (normal slides without any treatment) to completely fill your electrophoresis tank, so that none can move.
I agree with Emilie. Sounds like the slides moved during the electrophoresis due to the current. Immobilize your samples so they don't move and this should eliminate your problem.
If your direction of comet tail is diagonal then your slides are moved from their original place for sure. Add other slides to fill the tank and it will eliminate the problem. And you can remove the problem by adjusting the slide when you score it, rotate it in another direction. Only do this if you cannot repeat the experiment and all of your slides are diagonal.
Mone hoi, tor slide/ gel electrophoresis er somoy diagonally aligned hoye gechilo... amader BSc practical e erokom hoyechilo ekbar
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