SYBR Safe DNA Gel Stain—A Less Hazardous Alternative to EtBr

Studies proved that the transformation efficiency of DNA constructs stained with SYBR Safe DNA Gel Stain and visualized with Safe Imager Blue-Light Transilluminator is significantly higher than DNA constructs stained with ethidium bromide and visualized with UV transilluminator (Figure 1).

Furthermore, with increased duration of exposure to UV light, a 30-fold decline in the transformation efficiency of constructs stained with ethidium bromide was observed. In contrast, the duration of exposure to blue light did not affect the transformation efficiency of constructs stained with SYBR Safe DNA gel (Figure 1).

The cloning efficiency of bacteria transformed with PCR products stained with SYBR Safe stain and visualized with blue light illuminator remained at virtually 100% regardless of the duration of exposure to blue light (Figure 2). In contrast, the number of successfully transformed bacterial colonies using PCR products exposed to ethidium bromide/UV light was reduced by 80% when the UV exposure was 30 seconds compared to unexposed DNA or SYBR Safe-stained DNA.

SYBR Safe DNA Gel Stain is not classified as corrosive, ignitable, or reactive under the guidelines of the Environmental Protection Agency (EPA).

^‡ All tests were performed by AMEC Earth and Environmental San Diego Bioassay Laboratory, San Diego, CA.

SYBR Safe DNA Gel Stain does not differ significantly from 0.5X TBE buffer when tested according to National Pollutant Discharge Elimination System (NPDES) guidelines.

^††All tests were performed by Columbia Analytical Services, Inc., Kelso, WA. Methods used were as outlined in the Code of Federal Regulations (CFR) Title 40, Part 136.

SYBR Safe DNA Gel Stain in 0.5X TBE is indistinguishable from 0.5X TBE alone in terms of organic pollutant content. Both samples tested negative for the presence of an extensive array of organic compounds.

^‡‡ Samples analyzed were 0.5X TBE and 0.5X TBE + 1X SYBR Safe DNA gel stain; none of the compounds listed in the table were detected in either sample. Testing was performed by Columbia Analytical Services, Inc., Kelso, WA. Methods used were as outlined in the Code of Federal Regulations (CFR) Title 40, Part 136.

Some institutions and municipalities have approved the disposal of Invitrogen SYBR Safe DNA Gel Stain directly into their wastewater systems. However, disposal regulations vary. Please contact your safety office or local municipality for disposal guidelines.

In numerous tests carried out by independent, licensed testing laboratories, SYBR Safe DNA Gel Stain showed little or no genotoxicity and no acute toxicity. This stain is not classified as hazardous waste under US federal regulations. Nevertheless, please exercise common safe laboratory practices when using this reagent.

All Invitrogen SYBR dyes have similar spectral properties but have different chemical compositions. SYBR Safe DNA Gel Stain was specifically developed as a safer alternative to ethidium bromide. Invitrogen SYBR Green I Nucleic Acid Gel Stain is an ultrasensitive stain for dsDNA, and Invitrogen SYBR Green II RNA Gel Stain is a highly sensitive stain for RNA and ssDNA. All SYBR dyes are optimally excited by the Invitrogen Safe Imager Blue-Light Transilluminator.

Yes. Simply substitute a SYBR Safe DNA Gel Stain solution for the buffer when preparing the molten agarose. If using the 10,000X SYBR Safe DNA Gel Stain concentrate, dilute the concentrated stain 1:10,000 in agarose gel buffer (e.g., 1X TBE or 1X TAE) and add the buffer/stain solution to the powdered agarose. The agarose/SYBR Safe DNA Gel Stain mixture may be heated briefly in the microwave.

The SYBR Safe DNA Gel Stain content in a 1X solution is less than 1 ppm.

Water and 70% ethanol should get rid of most stains from spills. For persistent stains, bleach may be used. Use a handheld UV light in the darkroom to check for any remaining stain.

DNA stained with SYBR Safe DNA Gel Stain can be viewed using a blue light transilluminator such as Invitrogen’s Safe Imager instrument, or a standard UV transilluminator. If you plan to use the DNA for cloning, avoid exposing DNA stained with SYBR Safe DNA Gel Stain to UV light.

Unlike UV light, blue light causes minimal damage to DNA and is therefore safer for you and better for your DNA sample. Use of SYBR Safe DNA Gel Stain and the Safe Imager Blue-Light Transilluminator gives improved cloning efficiency over DNA stained with ethidium bromide and exposed to UV light.

Bands stained with SYBR Safe DNA Gel Stain are visible to the eye on a 300 nm transilluminator. However, optimum detection is obtained by photographing the gel using a UV-compatible emission filter with your CCD or film camera. UV bulbs may also emit some IR; if your camera lens is not specially coated to block IR, an IR-blocking filter is needed to prevent the appearance of faint images of the UV bulbs behind your gel. For optimum detection and improved safety, use the Safe Imager Blue-Light Transilluminator.

The Invitrogen SYBR Safe Photographic Filter mounts directly in front of the lens of any standard Polaroid® system, for those using Polaroid® B&W film (#667) to document their gels.

Please see "Filter recommendations for use with SYBR Safe DNA Gel Stain". Note that the excitation and emission spectra of SYBR Safe DNA Gel Stain are very similar to those of SYBR Green I, SYBR Green II, and Invitrogen SYBR Gold dyes, as well as fluorescein (FITC). Therefore, filters appropriate for these dyes can also be used. A camera filter is not required with the Safe Imager Blue-Light Transilluminator; the amber filter provided with the instrument serves this purpose.

Since there are so many different camera systems and emission filters, it is difficult to recommend specific photographic settings. Ideal exposure settings can be determined empirically. The manufacturer of the gel documentation system may be able to provide more detailed guidance.

Some ethidium bromide filters allow the transmission of all light above 500 nm. These filters (which are often yellow in color) and their associated camera settings can be used with SYBR Safe DNA Gel Stain, usually with only minor adjustments to the exposure or gain. Other ethidium bromide filters (often red in color) only transmit light around or above 600 nm; these filters and their associated camera settings are not suitable for use with SYBR Safe DNA Gel Stain.

SYBR Safe DNA Gel Stain has two main excitation peaks: in the UV region at 280 nm, and in the visible region at 502 nm. Thus, 254 nm or 300 nm UV excitation will work, as will 488 nm lasers, 470 nm LEDs, and broad blue excitation (such as the Safe Imager Blue-Light Transilluminator). Maximal excitation occurs at 502 nm; the Safe Imager Blue-Light Transilluminator is therefore the best choice for excitation of SYBR Safe DNA Gel Stain. The full excitation and emission spectra for SYBR Safe DNA Gel Stain are provided online and can also be found in the protocol provided with the stain.

Many whitening agents used in clothing, as well as some fungi and bacteria, fluoresce at the same wavelengths as SYBR Safe DNA Gel Stain. These contaminants within or on the surface of the gel may produce this “speckling.”

SYBR Safe DNA Gel Stain is compatible with all downstream applications we have tested so far, including excising PCR products from gels, gel purification, Invitrogen Gateway and TOPO cloning, and restriction enzyme cloning. If you have a unique application that works with SYBR Safe DNA Gel Stain, send us the details at techsupport@thermofisher.com.

Dilute SYBR Safe DNA Gel Stain concentrate 10,000-fold in TAE or TBE buffer prior to use. For most minigels, 50 mL of 1X stain is sufficient (e.g., dilute 5 µL of concentrate with 50 mL buffer). For larger gels, increase volumes proportionally, ensuring that the entire gel is fully immersed during staining.

SYBR Safe DNA Gel Stain yields the same sensitivity as ethidium bromide—roughly 500 pg/band in a minigel, for fragments larger than 200 bp viewed on a 300 nm transilluminator.

We strongly discourage the reuse of SYBR Safe DNA Gel Stain, as this practice significantly lowers sensitivity.

SYBR Safe DNA Gel Stain may be briefly microwaved with no loss of performance. However, we do not know the effect of repeated or long duration microwaving.

We recommend that SYBR Safe DNA Gel Stain be protected from light during storage and gel staining. However, it is sufficiently stable to withstand UV illumination for >30 minutes. Realistically, hours of constant UV or bright room light exposure are required to cause any significant loss of signal.

We have found a distinct advantage to using SYBR Safe DNA Gel Stain and non-UV blue light rather than ethidium bromide and UV when purifying DNA from gels for downstream use. The use of SYBR Safe DNA Gel Stain with non-UV blue light emitted by the Invitrogen E-Gel Imager System with Blue Light Base allows you to purify DNA with virtually no UV-induced nicking or crosslinking, resulting in dramatically increased cloning efficiencies.

Several Invitrogen E-Gel products are now available with SYBR Safe DNA Gel Stain. These gels can be used in the same manner as their ethidium bromide-containing counterparts. For more information on Invitrogen E-Gel pre-cast gels with SYBR Safe DNA Gel Stain, visit www.thermofisher.com/egels.

SYBR Safe DNA Gel Stain is easily removed from nucleic acids by ethanol precipitation.

Similar to ethidium bromide, SYBR Safe DNA Gel Stain runs in the opposite direction from that of the migrating DNA. This has no practical effect on the use of gels cast with SYBR Safe DNA Gel Stain, as only the very bottom of the gel will have a lower concentration of stain. How much of the gel will end up with a lower stain concentration is highly dependent on the agarose concentration, buffer used, and electrophoresis conditions including (voltage, wattage, and length of run, etc). This effect can be partially counteracted by adding SYBR Safe DNA Gel Stain to the running buffer.