|Ethidium bromide (EtBr) is intrinsically not as good a stain for the detection of DNA as the new SYBR dyes. This is partly due to the fact that the background fluorescence from unbound EtBr (i.e., fluorescence from EtBr in the agarose gel) is relatively high.
The background fluorescence is greatest when viewing EtBr-stained gels with a DR transilluminator. As a result, the DR is not as sensitive as a UV device for the detection of EtBr-stained DNA.
The background problem can be minimized by using a lower concentration of EtBr to stain the gel. We have found that staining a gel with an EtBr solution of 0.1 ug / mL (rather than the typical 1.0 ug / mL) significantly enhances the viewability of DNA bands. Using these conditions it is possible to see about 5 ng of dsDNA by eye and about 600 pg using a CCD or Polaroid camera. The best viewability by eye is achieved in a well darkened room and a few seconds should be allowed for the eyes to adjust to the low light levels.
Shown below are 2 images of a DNA gel stained with EtBr using our modified protocol and viewed on a DR transilluminator.
|Ethidium Bromide-stained DNA by Eye and Polaroid Film|
|Lambda DNA cut with StyI and SauI was fractionated on an agarose gel, stained with 0.1 ug/mL ethidium bromide and then viewed on a Dark Reader transilluminator. Image A is an attempt to approximate the DNA bands visible to the naked eye. Image B is an unaltered scan of a photograph taken with Polaroid 667 film. Notice that the detection limit on Polaroid film is much better than by eye.|
Clare Chemical Research