Hi, Coating slides for the comet assay is always an ungrateful task. There are probably no flaws in your agarose. I’ve experienced similar issues when trying to coat new slides. During my undergraduate studies in a lab with limited resources, we often reused old slides. After removing the agarose and samples, we boiled them, dried them, and cleaned them with ethanol. These reused slides tended to provide better agarose adhesion.

With new slides, I faced many difficulties. However, applying the agarose up to one-third of the frosted end of the slide helped improve adherence. You can also find slides designed specifically for comet assays, which have wells or a rectangular window in the center.

In our lab, we sometimes rely on what Brazilians call a “jeitinho brasileiro” or “gambiarra”. I used sandpaper to make my own comet assay slides by manually roughening a 0.5 cm border on all sides. This created a textured area that helped with agarose adhesion.

I hope this helps. Best regards.

Thank you so much, we will try this.

Dear Dr. Maria Perde,

Agarose Type I low EEO is generally not the preferred choice for comet assay.

Agarose with low EEO has a higher gelling temperature than low melting point agarose, which means it requires higher temperatures for gel formation, potentially damaging the DNA. Instead, you should use low melting point agarose which melts at a lower temperature (typically around 65°C) than standard agarose. This is crucial for the comet assay because it allows for gentle lysis of cells and prevents DNA damage during the electrophoresis step.

You may want to follow the below protocol.

You may pre-coat the slide with a layer of normal melting point agarose to help the cell-containing gel layer adhere. Normal melting point agarose has a higher melting point, making it suitable for forming a stable base layer on the slide that adheres well. It acts as a supportive layer on the slide. This layer helps the low melting point agarose, which contains the cells, to adhere better and prevents it from detaching during subsequent steps. 

So, normal melting point agarose is generally used to coat slides, while low melting point agarose is preferred for embedding cells. Agarose Type I Low EEO may not be optimal for the comet assay's specific requirement.

Regards,

Malcolm Nobre

Very well noted, Malcolm Nobre ! Indeed, the slides are coated with Normal Melting Point agarose. Low Melting Point agarose is used to apply the sample onto the pre-coated slides. Apologies for the confusion—all previous tips were intended to improve the adhesion of the Normal Melting Point agarose.

Thank you for your answers.

I have an agarose type 1 with low EEO (electroendoosmosis), I guess it was purchased accidentally. We have experience in the lab with comet assay and all the phases including coating slides but now, after a long period we didn’t perform it, this issue came up and it left is puzzle. The only thing we can blame is this agarose which we believe its normal melting.