GelStar DNA stain

'' ..detection limits with the same CCD system improved dramatically...when a Dark Reader blue light transilluminator was used in place of the standard UV transilluminator. ''
White and Wu (2001) Electrophoresis 22, 860-863

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.

Sensitive detection of GelStar using a Dark Reader

Left: Lambda DNA cut with StyI and SauI was subjected to electrophoresis in 0.7% agarose containing GelStar and photographed on a Dark Reader transilluminator using a Polaroid camera. The total DNA load, from left to right per lane was 15, 8, 4 and 2 ng. The marked band contains 28 pg of DNA.

Using GelStar ‘In-Gel’
GelStar works very well if the dye is added to the agarose before electrophoresis. The use of GelStar rather than SYBR Green as an in-gel stain has 2 main advantages:
The sensitivity of DNA detection achieved with GelStar in-gel is much higher than with SYBR Green. In fact, the sensitivity is almost the same as that achieved with post-staining - less than 20 pg of dsDNA using a CCD camera. (Pre-staining with SYBR Green has a sensitivity of about 300 pg of DNA.)
The presence of GelStar stain retards the migration behavior of DNA samples only slightly and in a predictable fashion - much like EtBr (see the Figures below) and unlike SYBR Green. Consequently, in-gel staining with GelStar is the recommended technique for the rapid and accurate determination of the sizes of fragments in DNA samples.

Adding GelStar to the agarose before running in a Dark Reader GelHead 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.

GelStar gel in real-time using a GelHead

Migration rates of DNA in the presence of GelStar stain

GelStar in-gel
EtBr post-stain
GelStar does not affect DNA migration rates
GelStar is much more sensitive than ethidium
DNA samples from plasmid preps. and restriction digests together with DNA standards (BioLine, Inc.) were subjected to electrophoresis either in a Reliant HS gel containing GelStar or in a dye-free agarose gel subsequently stained with EtBr. In both cases, the amounts of DNA loaded were the same. The results illustrate that the presence of GelStar does not affect the migration rate, and hence the size determination, of DNA fragments.
The migration distances of the DNA fragments in a molecular weight standard (BioLine, Inc.) were measured after electrophoresis either in a Reliant HS gel containing GelStar or in an dye-free agarose gel subsequently stained with EtBr. A careful examination of the data reveals that the DNA pre-stained GelStar actually behaves in a more 'linear' fashion than the unstained DNA.
The amount of DNA loaded onto the GelStar gel (above right) is clearly too high, resulting in smearing and the appearance of minor DNA contaminants. This is a direct consequence of the greatly enhanced sensitivity of GelStar versus EtBr for the detection of DNA. In practice, the amount of DNA loaded onto a GelStar gel can be reduced 5 - 10 fold. Reduced DNA loading levels can result in considerable savings in the cost of expensive reagents.

For example, the 2 x 5 uL of DNA standard typically used on a EtBr gel costs about $1 and the 2.5 U of Taq polymerase required in a typical 50 uL PCR reaction also costs around $1. Using GelStar, only 1 uL of DNA standard is required and PCR reaction volumes can be reduced to 10 uL. A detailed analysis of the costs of using the various DNA stains can be found at the `Gel Costs` link.

Of course, if one is attempting to locate minor DNA products in, for example, a 50 uL PCR reaction, then the increased sensitivity of GelStar will allow the visualization of DNA fragments that are simply undetectable with EtBr.
Clare Chemical Research

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