Dear colleagues, I have been doing some tests with blood cells from the tail of Mus musculus in the comet assay. I have only done the NC (distilled water or saline). However, I am having some problems with the nuclei, as they seem "blurry" and "diffuse". On other days, it is as if the DNA is being "lost" by the agarose. I have already varied the amount of blood with the LMP, but the problem continues.

Summary protocol:

- 0.5% LMP and 1.5% normal agarose

- I mix 10 µL of blood (collected with 50 uL of EDTA) with 100 µL of low melting agarose. I homogenize it and place it on two slides.

- After processing the material, I place it in fresh lysis solution for 24 hours, chilled.

- I let it rest for 20 min in cold electrophoresis buffer prepared on the day and with pH > 13. I start the electrophoresis: 25V and 300 mA for another 20 min (current controlled by the buffer volume).

- I neutralize with TRIS buffer for 15 min.

- I dry the slides, and then place them for 10 min in pure ethanol.

- I stain the slide the next day with 30 uL of DAPI (1 ug/ml) and place the coverslip.