Visualization: ethidium bromide (EtBr) and dyes

The most common dye used to make DNA or RNA bands visible for agarose gel electrophoresis is ethidium bromide, usually abbreviated as EtBr. It fluoresces under UV light when intercalated into the major groove of DNA (or RNA). By running DNA through an EtBr-treated gel and visualizing it with UV light, any band containing more than ~20 ng DNA becomes distinctly visible. EtBr is a known mutagen, and safer alternatives are available, such as GelRed, which binds to the minor groove. Even short exposure of nucleic acids to UV light causes significant damage to the sample. UV damage to the sample will reduce the efficiency of subsequent manipulation of the sample, such as ligation and cloning. If the DNA is to be used after separation on the agarose gel, it is best to avoid exposure to UV light by using a blue light excitation source such as the XcitaBlue UV to blue light conversion screen from Bio-Rad or Dark Reader from Clare Chemicals. A blue excitable stain is required, such as one of the SYBR Green or GelGreen stains. Blue light is also better for visualization since it is safer than UV (eye-protection is not such a critical requirement) and passes through transparent plastic and glass. This means that the staining will be brighter even if the excitation light goes through glass or plastic gel platforms. SYBR Green I is another dsDNA stain, produced by Invitrogen. It is more expensive, but 25 times more sensitive, and possibly safer than EtBr, though there is no data addressing its mutagenicity or toxicity in humans. SYBR Safe is a variant of SYBR Green that has been shown to have low enough levels of mutagenicity and toxicity to be deemed nonhazardous waste under U.S. Federal regulations. It has similar sensitivity levels to EtBr, but, like SYBR Green, is significantly more expensive. In countries where safe disposal of hazardous waste is mandatory, the costs of EtBr disposal can easily outstrip the initial price difference, however. Since EtBr stained DNA is not visible in natural light, scientists mix DNA with negatively charged loading buffers before adding the mixture to the gel. Loading buffers are useful because they are visible in natural light (as opposed to UV light for EtBr stained DNA), and they co-sediment with DNA (meaning they move at the same speed as DNA of a certain length). Xylene cyanol and Bromophenol blue are common dyes found in loading buffers; they run about the same speed as DNA fragments that are 5000 bp and 300 bp in length respectively, but the precise position varies with percentage of the gel. Other less frequently used progress markers are Cresol Red and Orange G which run at about 125 bp and 50 bp, respectively.

Added: July 12th 2011
Reviewer: John Adamas
Score: 8  
Hits: 388
Language: english

  

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