I've performed a COMET assay multiple times, improving on the procedure each time and re-making my solutions, but continue to get the same inconclusive results. My supervisor and I both tried refocusing the microscope but it does not appear to be able to focus on anything beyond what is imaged. I am trying to interpret what I am seeing, and where I went wrong in my COMET assay. It does look like a few COMET-like forms are in some of the photos, but it seems wrong because they are in all different directions.
Any thoughts, suggestions, or advice would be greatly appreciated!
Thank you!
I've added a simplified version of my procedure below:
Day 1:
Day 2:
Day 3:
Day 4:
I had this once - comets in different directions. When you place slides in the electrophoresis chamber, ensure all slides are orientated in the same direction. For example the frosted end of each slide facing right. My chamber can hold a total of 12 slides in two rows of 6 each. In both rows, I place the slides such that the frosted end of all slides is to the right.
Once I corrected for this, I have had not had this issue again. Hope this helps.
Dear Brenna Hay,
We perform Comet assay differently (if you want zou can check here - Article Klebsiella oxytoca enterotoxins tilimycin and tilivalline ha...
But I immediately can give some suggestions :
1)It is no need to leave slides in lysis solution overnight just 1h should be okay.
2)Ratio between cells and LMP should be from 1 to 6 till 1 to 8.
3) It is also no need to use cover slips, with this one you still can damage cells.
4)Instead of leaving slides for 20 min. you can use Alkaline solution (just 3 g of NaOH in 100 ml of water)
5) again it is no need to neutralize electrophoresis solution, just wash slide with water 3 times for 2-5 min.
6) All, Slides should be fixed during electrophoresis.
Thank you so much, I'm giving it another try in the next week or so and hopefully get better results!
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