SYBR Green

SYBR Green I stain was the first of the new generation of DNA stains introduced by Molecular Probes in 1995 and since then has been quietly revolutionizing the detection of DNA in gels. It has also found numerous other applications including the the quantitation of PCR.
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 less than 100 pg of SYBR Green-stained DNA by eye and tens of picograms using a CCD system.

DNA gel stained with SYBR Green and viewed on a Dark Reader

Apart from its superior sensitivity, SYBR Green stain has other advantages over EtBr:
- It is much less mutagenic,
- It can be added directly to the DNA sample before electrophoresis. (See below for a discussion of the advantages of using SYBR Green as a pre-stain.)

SYBR Green is much less Mutagenic than Ethidium

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.

SYBR Green is much less mutagenic than Ethidium
Using SYBR Green as a Pre-stain
Adding SYBR Green directly to DNA samples before electrophoresis has a number of major advantages:

- DNA bands can be directly visualized as they migrate through the gel using the Dark Reader GelHead as shown on the right. An electrophoresis run can be halted as soon as the desired DNA bands are separated - often within 30 minutes.
SYBR Green can be used as a 'pre-stain'

- Unlike ethidium bromide (which when used as a pre-stain must be added to the gel and the running buffer) SYBR Green need only be added to the DNA samples. This drastically reduces the amount of dye required and virtually eliminates the risk of toxic spills.
- By avoiding the post-staining step, electrophoresis results are obtained immediately.
- SYBR Green is very economical to use as a pre-stain (about 0.3 cents per sample).
Tips on Using SYBR Green
1. For best results when pre-staining the DNA sample, use a dilution of 1:1,000 of SYBR Green. In the CCR lab, we keep a stock of 1:100 SYBR Green in DMSO and add 1 uL of this for every 9 uL of DNA. The DMSO does not appear to affect the DNA.
2. Run the gel in less than 2 hours. During overnight runs the SYBR dye tends to dissociate from the DNA resulting in smeared bands.
3. SYBR Green slightly retards the migration of DNA standards. However, there is still a predictable relationship between size and mobility. When used with DNA samples generated by typical laboratory procedures such as PCR and plasmid preps., the migration of DNA fragments becomes much more unpredictable. For the rapid and accurate determination of DNA fragment sizes, the use of GelStar as an in-agarose stain is recommended.
4. Using SYBR Green as a pre-stain, the sensitivity of detection is not as high as with post-staining in a bath. It is possible to see about 300 pg of dsDNA by eye.
5. There is one report (Miller et al, BioTechniques 27, 34 -36, 1999) that at higher DNA concentrations (over 50 ng per band), a more significant alteration of the mobility of pre-stained DNA is observed. (A similar phenomenon occurs with ethidium bromide.)
Clare Chemical Research

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