comet assay - DNA damage in negative controls

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rachan673's picture
comet assay - DNA damage in negative controls

hi everyone

i'm involved in performing comet assay for drug molecules. but the problem is that negative controls are also showing comets like that produced by positive agent. i tried many times but without any success.

could anyone please help me out of this.
all sugestions and advices are most welcome.

thank you!

g a
g a's picture
Hi Rachan673

Hi Rachan673

Comet Assay detects the nuclear damage.... so Is it quite likely that either your drug doesnt induce DNA damage. Have you checked that possibility.

Secondly, the technique is quite sensitive and the damage usually detected are single strand breaks and double strand breaks. It is sometimes stated that alkaline conditions and complete denaturating of the DNA is necessary to detect single strand breaks. However this is not true, both single- and double strand breaks are also detected in neutral conditions. In alkaline conditions, however, additional DNA structures are etected as DNA damage: AP sites (abasic sites missing either a pyrimidine or purine nucleotide) and sites where excision repair is taking place.

Check for that possibility as well