Detect less than 100 pg of DNA
Using a unique combination of filters, the Dark Reader is more sensitive than a UV transilluminator for the detection of many stains. For example, using SYBR Gold stain it is possible to see, by eye, less than 100 pg of DNA.
Viridi Easy + Viridi Vivid
SYBR Green
SYBR Green I stain was the first of the new generation of DNA stains introduced by Molecular Probes in 1995. The fluorescence intensity of SYBR Green is enhanced over 100-fold on binding to DNA which results in bright fluorescent DNA bands against a very dark background. Using a Dark Reader transilluminator, it is possible to detect about a 100 pg of SYBR Green-stained DNA by eye and tens of picograms using a digital camera.
Apart from its superior sensitivity, SYBR Green stain is much less mutagenic than EtBr. It is not recommended to add SYBR Green to the agarose because DNA migration rates are unpredictable.
Researchers at Molecular Probes compared the mutagenicity of SYBR Green I stain with that of ethidium bromide in Salmonella / mammalian microsome reverse mutation assays (Ames tests). They concluded that SYBR Green I stain is only a weak mutagen and appears to be much less mutagenic than ethidium bromide. (Singer et al., Mutat. Res. 1999, 439: 37- 47.) One possible explanation for this is that SYBR Green does not intercalate between the DNA bases and if it does, that its presence does not give rise to point mutations at a high frequency.
GelStar
GelStar stain (Lonza Bioscience) can be used for the highly sensitive detection of dsDNA, ssDNA, oligonucleotides and RNA in gels. Our results show that the detection limit of dsDNA stained with GelStar and viewed using a Dark Reader is comparable to that of SYBR Green and SYBR Gold stains.
Adding GelStar to the agarose before running the gel allows the DNA migration to be monitored during electrophoresis. The time savings can be significant – staining time is eliminated plus the gel only needs to be run until the bands of interest are separated – about 40 min, depending on the complexity of the sample. The total time saved can be over 1.5 hours, start to finish.
Ethidium Bromide
Ethidium bromide (EtBr) is intrinsically not as good a stain for the detection of DNA as the new stains. 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.
In addition EtBr is better excited by UV light than blue light and consequently will always look better on a UV transilluminator. It is possible to see 5 – 10 ng of DNA using a Dark Reader and many of our customers do continue to use ethidium.
SYBR Gold
SYBR Gold is the most sensitive of the dyes we have tested for the direct visual detection of dsDNA and it is possible to see less than 100 pg of dsDNA by eye on a Dark Reader transilluminator. Using a digital camera it is possible to detect 10 – 25 pg.
SYBR Safe
‘Safe’ is the latest addition to the SYBR® stain family and has a number of very attractive benefits:
- Increased safety – Data released by Molecular Probes show that SYBR Safe stain is negative in 3 different mammalian cell-based assays for genotoxicity and exhibits low mutagenicity compared to EtBr in Ames tests. Furthermore, SYBR safe has been extensively tested for environmental safety and is NOT classified as hazardous waste under U.S. Federal regulations.
- Flexible usage – SYBR Safe can be used either to stain a gel after running or, for the fastest results, it can be used as an‘ in-agarose’ stain.
- High stability – Safe is stable for over 6 months in aqueous solution and is also stable to the heating involved in preparing molten agarose.
SYBR Safe, however, will only detect about 2 ng of DNA and is
one of the least sensitive of the new generation of DNA stains.
GelGreen
GelGreen™ from Biotium, Inc has a number of attractive features:
- GelGreen can be used in agarose
- Is stable in aqueous solution
- Shows no mutagenic effects in Ames tests
- Is more sensitive than SYBR Safe
