The attached pictures are all cells that have undergone the same treatment in the same agarose gel. Why is there so much variability?
My protocol is as follows:
-Resuspend pellet in PBS.
-60min in Lysis Buffer at 4 degrees Celsius in the dark
-30 min in Alkaline Electrophoresis Buffer at 4 degrees Celsius in the dark
-40 min electrophoresis at 1volt/cm, 300mA current
-Dehydrate with 70% ethanol for 5 minutes, airdry overnight
-Stain with DAPI
Thank you for your quick response Harald!
My positive control is radiation (10 Gy) only. I've been trying to use H2O2 as another positive control but that hasn't been successful.
I recently switched to using a specialized comet assay slide (OxiSelect 3-well), which are what the photos above are from.
The photos in this post are from when I used conventional microscope slides coated with NMA. (I switched because I kept struggling to keep the LMPA on the slide throughout the steps).
I'm not sure why the results are so different. The 'old' comets appear much more consistent with a regular "cloud tail", while these new ones have a bit of a halo, the nucleus is hard to see and the tail pieces are really distinct.
It looks to me like your cells are in different depths in the agarose so they are not all in focus at the same time. Try zooming out with your camera and see if you get better resolution with a larger field of view.
Dear Morla Phantastic,
Your positive control seems totally fine.
In our case we also have huge variability in treatment between cells.
Article Klebsiella oxytoca enterotoxins tilimycin and tilivalline ha...
This can be explained :
1) All cells react differently on treatment
2) Your treatment need different time point ?(depends on cell line)
3) Your drug cann´t be transferred so rapidly ... etc.
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining