Advantages
No safety risks
We don't like UV exposure...
Great cloning efficiency
Your plasmids will thank you!
No DNA damage
Mutation-free fluorescence
Cut bleaching
Fluorophores that keep going
See through walls
Plastic doesn't get in our way
Is UV causing problems?
Decreased efficiency – UV transilluminators typically output light around 300 nm – well removed from the absorption maxima of most common dyes.
Occasional Sunburn? – Many users of UV transilluminators have experienced, at one time or another, either a mild case of sun-burn or `spots before the eyes` as a result of spending too long either examining a gel or cutting out bands. The potentially harmful effects of shorter wavelength light (less than 400 nm) are well documented and were even the subject of a report from the Council on Scientific Affairs of the American Medical Association .
“high-intensity UV radiation can cause erythema, degenerative and neoplastic changes in the skin, retinal damage and cataracts, and modification of the immunologic system of the skin.”
Council on Scientific Affairs, JAMA, 1989, 262, 380-384
No UV output = No DNA damage
DNA undergoes a number of reactions when exposed to UV light. Because the Dark Reader does not emit any UV light, the extent of DNA damage is drastically reduced compared to the damage produced by a UV device.
100 ng of supercoiled (sc) plasmid was placed on either a Dark Reader (DR) or a 312 nm UV transilluminator (UV) for various times. The DNA was then digested with T4 endonuclease V, which excises T:T dimers, and run on an agarose gel and viewed. It is clear that as little as a 5 sec exposure to UV light is sufficient to convert almost 100% of the plasmid into the relaxed (rx) form and after 300 sec the DNA is completely fragmented. In contrast, a 300 sec exposure on the Dark Reader resulted in no detectable damage to the plasmid.
We worked hard to find an alternative!
The Dark Reader Uses Blue Light
The light sources in Dark Reader devices generate maximum light output between 400 and 500 nm – close to where dyes such as SYBR Green, SYPRO Orange, eosin, fluorescein and ethidium bromide are excited. UV transilluminators, on the other hand, typically output light around 300 nm – well removed from the absorption maxima of most common dyes.
The Dark Reader uses 2 Filters to reveal Fluorescence
If visible blue light is used for excitation of a fluorophor, any fluorescence from the sample is not directly detectable by the naked eye due to the large amount of incident light from the light source itself that reaches the observer.
The Dark Reader achieves the removal of incident light in 2 steps. The first filter is between the light source and the DNA. This removes any green and red components from the lamp and allows through to the DNA only blue excitation light.
A second filter is placed between the DNA and observer that removes the blue incident light but allows passage of the red and green fluorescent components.
How it works
Many fluorophors actually absorb visible light. The excitation maxima for many popular dyes, including SYBR Green and red-shifted GFPs, are between 400 and 500 nm – not in the UV (see Fig.1). These wavelengths correspond to blue-green light which is well within the visible spectrum.
The good things it will do for you
Maximum cloning efficiency
Numerous reports (1-8) show that exposure of DNA samples to UV radiation has a detrimental impact on downstream protocols. Transformation, transcription and PCR efficiencies are all reduced by 2-3 orders of magnitude when DNA samples are exposed to UV light – even for the brief period of time that it takes to excise a band from a gel. Also, the use of UV-exposed DNA templates can result in reduced fidelity and incorrect DNA replication. as well as impair the biological integrity of proteins encoded by the exposed DNA.
Described below are the results from experiments performed by various independent scientists showing the dramatic improvements (102 – 104 !) that can be achieved when DNA samples are viewed on a Dark Reader rather than a UV transilluminator.
Method
In the experiments conducted by researchers at Epicentre, T7 DNA or wheat germ chromosomal DNA was incubated with End-Repair Enzyme Mix and the DNA fragments were resolved by electrophoresis and stained with SYBR Gold. During excision of the DNA bands from the gel, the DNA was exposed to either Dark Reader (DR) light or UV light (302 nm) for 30 seconds. The purified DNA was then ligated to the pWEB Cosmid Vector and the ligated DNA was packaged into lambda particles using the MaxPlax Lambda Packaging Extracts. EPI305 plating cells were then transfected, plated, and incubated overnight. Either plaques (for T7 DNA), or colonies (for wheat germ DNA) were counted.
Results: Wheat Germ DNA
Exposure of the wheat germ DNA to 302 nm UV light for 30 seconds had a large impact on the cloning efficiency. The plating efficiency of the UV-exposed DNA was 2 x 104 cfu/ug DNA. In contrast, the plating efficiency of the Dark Reader (DR) – exposed DNA was 85-fold higher, with 1.7 x 106 cfu/ug DNA. These results demonstrate that UV-exposed DNA is significantly compromised in its ability to function well in such applications as the development of genomic libraries. For library construction an 85-fold reduction in cloning efficiency can be very significant, particularly for larger genomes.
Results: T7 Phage DNA
Viral infection with T7 phage requires that the cloned T7 DNA in each cosmid express the gene products, in active form, necessary for T7 phage production. DNA recovered from a gel visualized on a Dark Reader transilluminator produced a 220-fold greater number of plaques (2.2 x 106 pfu/ug DNA) than DNA recovered from a gel exposed to light from a UV transilluminator (1 x 104 pfu/ug DNA). These results are even more dramatic than those obtained with the wheat germ DNA. This clearly indicates that, in addition to affecting ligation and transformation efficiencies, the 30-second UV exposure compromised the activity and function of one or more of the gene products necessary for T7 phage production.
Dr. A. Michael Chin, the founder of Sequetech, tested a Dark Reader for visualizing DNA templates to be used in PCR reactions. Here are his notes:
Method
1. Ran some DNA fragments through an agarose gel in duplicate.
2. Cut the gel in half and stained one half with SYBR Gold and the other with ethidium bromide.
3. De-stained the ethidium bromide stained half. It was not necessary to de-stain the SYBR Gold half, saving some time.
4. Visualized the SYBR Gold half on the Dark Reader, took a picture and cut out the band I wanted.
5. Visualized the ethidium bromide half on a standard UV transilluminator, took a picture and cut out the band I wanted.
6. Purified both bands away from the agarose.
7. Performed PCR amplification of both purified DNA fragments. Started the amplifications with undiluted, 1/100 diluted and 1/10,000 diluted fragments for a total of 6 amplifications.
8. Ran all amplifications through an agarose gel and visualized them.
Results
All 3 of the Dark Reader amplifications maxed out the reactions while only the undiluted amplification of the UV-exposed template maxed out the reaction.
Conclusion
The DNA which was isolated using the Dark Reader was at least 10,000 times as amplifiable as the DNA isolated using ethidium bromide and a standard UV transilluminator. Since all of the Dark Reader amplifications were maxed out, they may be more that 10,000 times as amplifiable as the ethidium bromide/UV DNA.
1. Brunk, C.F. and L. Simpson. 1977. Comparison of various ultraviolet sources for fluorescent detection of ethidium bromide-DNA complexes in polyacrylamide gels. Anal. Biochem. 82:455-462.
2. Hartman, P. S. 1991. Transillumination can profoundly reduce transformation frequencies. BioTechniques 11:747-748.
3. Daum, H.A., H.G. White, C.M. Seidell and P.A. Johnson. 1991. Cloning, restriction digestion and DNA labeling of large DNA fragments in the presence of remelted SeaPlaque GTG agarose gels. BioTechniques 11:784-790.
4. Cariello, N. F., P. Keohavong, B.J.S. Sanderson and W.G. Thilly. 1988. DNA damage produced by ethidium bromide staining and exposure to ultraviolet light. Nuc. Acids Res. 16:4157.
5. Grundemann, D. and E. Schomig. 1996. Protection of DNA during preparative agarose gel electrophoresis against damage induced by ultraviolet light. BioTechniques 21:898-903.
6. Paabo, S., D.M. Irwin and A.C. Wilson. 1990. DNA damage promotes jumping between templates during enzymatic amplification. J. Biol. Chem. 265:4718-4721.
7. Hoffman, L. 1996. T4 endonuclease V detects UV transilluminator damage to DNA in agarose gels. Epicentre Forum 3:4-5.Y
8. Jiang, C Ke, PA Mieczkowski, and PE Marszalek 2007 Detecting Ultraviolet Damage in Single DNA Molecules by Atomic Force Microscopy Biophysical Journal 93:1758-1767
See Through Walls
Maybe not brick walls; but certainly test-tube walls, 96-well plate walls, gel plate walls….. all sorts of plastic and glass walls. There are 2 reasons for the superiority of the Dark Reader compared to UV:
No blocked light
Dark Reader blue light easily passes through transparent materials. With a Dark Reader hand lamp, EGFP can easily seen migrating down a gel!
No background
Many plastics fluoresce when exposed to UV light. In the case of UV irradiation, the fluorescence from the sample is difficult to separate from the fluorescence from the tube itself. Using a Dark Reader, there is essentially no fluorescence from the tube and the fluorescein sample is clearly visible.
Reduce Photo-Bleaching
The photo-bleaching of fluorophors upon exposure to light can become a significant problem, particularly when the experimental protocol is prolonged. This situation arises, for example, when proteins are being isolated from 2-D electrophoresis gels for downstream analysis. Clearly, if photobleaching can be minimized then the usable life of a gel can be extended accordingly, without the need to re-stain the gel.
To determine the extent of photobleaching that occurs upon exposure of SYPRO Orange- stained proteins to DR and UV light, samples were variously exposed for 8 minutes on either a DR or a 312 nm UV transilluminator. The results below show that UV exposure causes a ~40% decrease in the fluorescence intensity of the protein bands. Interestingly, some proteins appeared to be more significantly affected than others and were almost undetectable after 8 minutes of UV exposure. The DR exposure, on the other hand, resulted in only a ~10% or less decrease in band intensity, indicating that the DR transilluminator is a more appropriate device for procedures that require extended exposure to exciting light.
The photo-bleaching of SYPRO Orange-stained proteins by UV and DR light was measured. An SDS polyacrylamide gel was loaded with 3 aliquots of protein molecular weight standards (15 ng per band), subjected to electrophoresis and then stained with SYPRO Orange. Two of the protein lanes were cut out from the gel and exposed on either a 312 nm UV transilluminator (UV) or a DR transilluminator (DR) for 8 minutes. The protein lanes were then all photographed together on a DR transilluminator and the intensities of the fluorescent bands measured using IGOR 4.0 image analysis software.
FAQ
Doesn’t get any safer. The Dark Reader optical system contains less UV light than the ambient fluorescent lighting used in most offices and laboratories. Because the Dark Reader transilluminator emits almost immeasurably low levels of light below 400 nm, there is essentially zero risk of UV radiation causing eye or skin damage, making it much safer to use than a traditional UV transilluminator.
Summary of most common stains:
| Viridi Vivid, SYBR Gold | < 100pg |
|---|---|
| SYBR Green , GelStar | ~ 200 pg |
| GelGreen | ~ 300pg |
| SYBR Safe | < 1ng |
| EtBr | > 5ng |
The glasses are not for eye protection. (Indeed, do not use them with a UV unit because they do NOT provide protection against UV !)
The light from the Dark Reader is all visible blue. Of course, it is fairly bright so you should not look directly at it for too long (without the glasses or amber screen in place) – just like any other visible light source – but there is essentially zero UV in it. The function of the glasses is optical – they prevent the blue excitation light from swamping the much fainter fluorescent light from the sample. They have exactly the same optical properties as the amber screen and DR camera filters. The glasses are especially useful for when you are cutting bands out of a gel. They save you trying to work around the amber screen
