Question on Ethidium Bromide Gels - (Jun/24/2006 )
I have a technical question about the detection of PCR produced DNA on ethidium Bromide gels. In our lab, we add EtBr directly to the gel before solidification, run the gel, and detect DNA bands after with a UV light source and a polaroid camera setup. I am given to understand that EtBr will bind in between the bases of the DNA and that is why the bands show up under UV light. My question is, is if EtBr is actually in the gel, why the whole gel does not light up under the UV light? Also, does the EtBr bind to the DNA as it moves through the gel, and if so, would this mean that shorter bands would be brighter as they move further through the gel, picking up more EtBr as they pass through? I don't have much experience with this, but I would like to have these issues clarified or explained if anyone could help.
EtBr is an Intercalating Agent, meaning it wedges itself into the grooves of DNA and stays there. More base pairs mean more gooves, which in turn means more EtBr can insert itself. The EtBr has a background fluorescence and you see the bands of DNA simplu because there's more EtBr where the DNA is. Moreover, the bigger the band, the more EtBr is incorporated, the more intense the band. The EtBr runs to the opposite direction as the DNA: it happens that when the DNA goes to the end of the gel, the EtBr is not there anymore and it's more difficult for you to see the band
I agree with dnafactory. When one runs a gel with a sample DNA, the band is brighter when observed intially than when compared at a later time point.