There is no point in running comet assay at 45V. One must run it slow at 21mA and constant current of 300mA for 20-30min. Your positive control seems fine. It seems that the pH of your buffer is not proper or the running buffer has not been made properly. Please refer to my papers here and follow the same.

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Hi

main issue is when you prepare the buffer.

for reaching to right pH you have to consider the amount of free ions.

when it was ok the voltage and A that protocols say comes true.

Tasaduq Hussain and Alireza thank you for your suggestions.

As far as I understand from your comments, my problem is related to the running buffer.

For electrophoresis buffer I firstly prepare stock solutions:

10 N NaOH (200 g/500 mL dH2O)

200 mM EDTA (14.89 g/200 mL dH2O, pH 10)

While the slides are in the lysis solution I prepare 1X buffer from stock solutions as 300 mM NaOH / 1 mM EDTA ph>13.

I will try the assay one more time by preparing the solutions more carefully.

Dear Nebahat Yıldızbayrak,

Greetings!!

Following are my suggestions to your queries:

Query 1: Adjust the current to 300 mA by either lowering or raising the buffer level.

Query 2: Instead of 1.5 % Normal Melting Point agarose, you can prepare your base slides in 1 % Normal Melting Point agarose. Care should be taken that the agarose is spread uniformly in the slide.

Also, you should add a final third agarose layer (80 μL Low melting point agarose) to the slide.

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