I have been trying to standardize comet assay for sometime now but I just have not been able to see the comets whatsoever even in my positive control which are cells treated with hydrogen peroxide. I have been using a concentration of about 60-50 micromolar to induce DNA damage (for 30 mins).

1. Mix 1% LMP agarose with the cell suspension by pipetting gently up and down while avoiding the introduction of air bubbles (take 45 μL of the cell suspension (~1 × 105 cells/mL) and mix with 105 μL of 1% LMP agarose at 37 °C, resulting in a final concentration of 0.7% LMP agarose). Transfer two 40–75 μL drops to each microscope slide precoated with 1% NMP agarose. Allow to solidify at 4˚ C and perform standard lysis.

2. Lysis (2.5 M NaCl, 0.1 M Na2EDTA and 10 mM Trizma base, pH 10 (with 10 M NaOH). Before use, add 1 mL of Triton X-100 per 100 mL) at 4˚C overnight.

3. DNA unwinding (alkaline buffer solution - 0.3 M NaOH and 1 mM Na2EDTA) at 4˚ C for 1 hour.

4. Electrophoresis using the same buffer for 30 mins at 1V/cm (28V) at 4˚ C (in ice).

5. Neutralization with 1X PBS for 10 mins. Dehydrate the slides with 70% ethanol for 10 mins. Allow to airdry.

6. Staining with 1µg/ml DAPI for 10 mins. Wash the slides with dH20 to remove the excess stain. Airdry the slides and observe under fluorescence microscope.

My concern is also that I observe the nuclear material in very poor quality. I don't know if it because of the agarose but I have attached the image. I just can't seem to focus it better. I am using Zeis fluorescence microscope at 40X magnification. What can I do? Please help a troubled researcher out :/