I did comet assay with goldfish in my master's degree but now I am experiencing such a problem. has anyone encountered such a problem before? the only difference from my other experiments is that the LMAs have been in the ependorf in the refrigerator for a while and while taking blood, I cut the anal fin and put it on the pbs from the cut point and let the blood flow into the pbs. there was no difference other than these two.
Thank you in advance for your assistance.
How long period did you store the samples.
If the storage period is too long, there is a possibility that the DNA may degrade.
The pH of the reagents used is also important. For example, is the pH of the mincing buffer strictly adjusted? If the pH is too high, the DNA will degrade.
I kept the solution at room temperature during lysis, not on ice. I will repeat the experiment and share my results with you. thank you very much for your advice.
The pH of all the solutions should be checked, especially the pH of PBS; a freshly prepared lysing solution should be used; and it should be cold or on ice during the lysing period. You can collect the blood by puncturing the dorsal aorta posterior to the anus with the help of a heparinized capillary.
I hope it will help you.
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