Comet assay problems

4 posts / 0 new
Last post
Rawiwan
Rawiwan's picture
Comet assay problems

I try to do the comet assay with human lymphocytes, the problem is when i treated the cell lymphocytes with the negative control; distilled water, its generated the comet tail as same in the cell treated with H2O2.

Please give me a suggestion, how to solve this problem?

Thank you

RW

FLHPI
FLHPI's picture
Hello, it's most likely that

Hello, it's most likely that some DNA damage is occuring to your negative control cells. 
Try the following:
Check morphology of cells to ensure healthy appearance before sample preparation.
Handle cells gently to avoid physical damage.
Ensure electrophoresis buffer solution remains below 20°C.
Keep cells on ice as much as possible and prepare cell samples immediately before combining with molten LMAgarose.
If an enzyme disaggregation method is used, try reducing exposure time or enzyme concentration.
I got this information from this comet assay trouble shooting guide:
http://www.scorecomets.com/about-the-comet-assay/troubleshooting-guide

You might find it useful in the future?
Best wishes.

Rawiwan
Rawiwan's picture
Thank you so much for your

Thank you so much for your suggest.
After i checked the morphology of cells, i found that many cells had damaged.
So, i will  isolate the lymphocytes and try to do it again with followig your suggestions.

Thank you so much :)

Rawiwan

Yeshani Samarakoon
Yeshani  Samarakoon's picture
In alkaline comet assay , if

In alkaline comet assay , if the pH of the unwinding/electrophoresis buffer is very high (pH over 14) does it have any effect on comet formation?