Hello!
I'm still trying to get the assay to work, first time to do it. Followed the standard Singh et all 1988 protocol for alkaline comet assay. I used different drugs (0.1%H2O2 and 18ug/ml Bleomycin for an hour). Lysis for 1.5 hours and electrophoresis for 20 mins, after I allow denaturation for 20 mins in the tank.
I'm thinking maybe it's inefficient lysis (these are U2OS cells) such that DNA is not freed from the proteins and thus migration is affected (do i just increase time or is there a tip here?, or the agarose. I think agarose is too much. This is why i would like advice on how you prepare your slides and cell+LMPagarose suspension, and how many layers of agarose do you use for this assay, 1, 2 or 3? i think the agarose could be causing this blurry staining/poor migration of DNA.
(The attached pics , cells could not be more focused using adjustment they just seem blurry at any level and i think it's because of too much agarose the cells are not close enough to the coverslip where the light source illuminates the cells/fluoresces the DNA)
Thanks!
What were youur control samples like - Positive and Negative controls...?
Your hydrogen peroxide must be made Fresh within 30 minutes of use to preserve its 'damaging' abilities. Once made store at 4 degrees in a dark bottle. If there is no change...
Check your Lysis Solution (1% sodium lauryl sarcosinate) has not precipitated at 4 degrees. It may be the damaged DNA can't get out of the cell. If there is still no improvement...
Possibly try increasing the exposure time on the CONTROL samples ONLY to begin with. Negative control with comet tails means you have gone overboard on either damage or lysis steps
Finally as a last resort try increasing the hydrogen peroxide concentration again in Control samples only to begin with
Hello Caroline, Thank you for helping out, well yes it could be our H2O2 is old, but i used another positive drug of Bleomycin, that's what made me think there's something wrongwith the assay itself (the way i do it) not the drugs being old. I see, but i didn't use the detergent sarkosyl (i just used Triton X, not both, which is shown in several protocols.. do you use both? Triton and Sarkosyl? I think i will consider using sarkosyl as it's used in a clear comet assay protocol published in Nature Thank you!
Hello Harald, Thank you for your input, yes i used Triton X 100 (freshly prepared buffers), aha-okay i will try increasing electrophoresis time next time.. how long do you usually run? yes i dried slides in absolute ethanol before staining
Perhaps before you do any more have a read up on variations of the method
http://www.nature.com/protocolexchange/protocols/5207#/procedure
and this is a powerpoint summary of comet assay
http://environmentaltoxicology.conferenceseries.com/ppt/2017/recommendations-to-enhance-reproducibility-and-reliability-in-comet-assay
Are you using a kit ?
We basically use Dhawans technique but add sodium sarcosinmate to the lysis solution.On the slide coated with nmpagarose we add 1 layer of cells and lmpagarose
Dear Salma,
Send feedback as I prepare or slide with agarose and lysis phase. Best regards
It may be worth increasing the time the gels are in lysis. We keep our slides in lysis for a minimum of 18 hours
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