LIFE TECHNOLOGIES SAFE IMAGER 2.0 BLUE-LIGHT TRA

Catalog No. G6600 Supplier LIFE TECHNOLOGIES Supplier No. G6600

Frequently Asked Questions (FAQs)

Which direction does the SYBR Safe dye run during electrophoresis?

Similarly to ethidium bromide, SYBR Safe DNA Gel Stain runs in the opposite direction 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. This effect can be partially counteracted by staining the gel with SYBR Safe DNA Gel Stain after electrophoresis. Solutions of dye should not be added to the running buffer as this can cause breakdown of the dye at the electrodes and release toxic volatile compounds into the air.

How do I view DNA stained with SYBR Safe DNA Gel Stain?

DNA stained with SYBR Safe DNA Gel Stain can be viewed using a blue-light transilluminator such as our Safe Imager 2.0 Blue-Light Transilluminator 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.

What are the specifications for your Safe Imager 2.0 Blue-Light Transilluminator (Cat. No. G6600)?

Please see the instrument specifications below:

- Instrument dimensions: 195 - 325 - 65 mm (11.6 - 12.8 - 2.6 in)
- Viewing surface dimensions: 190 - 190 mm (7.5 - 7.5 in)
- Light source: light emitting diodes (LED) producing a narrow emission peak centered at ~470 nm
- LED life: 50,000 hours
- Complies with the European Community Safety requirements
- Contains Class 1 LED products
- Included accessories: amber filter unit, viewing glasses, and international power cord

What is the difference between the E-Gel Safe Imager Real-Time Transilluminator (Cat. No. G6500) and the Safe Imager 2.0 Blue-Light Transilluminator (Cat. No. G6600)?

The E-Gel Safe Imager Real-Time Transilluminator docks onto the E-Gel iBase Power System and allows real-time imaging of the migration of DNA/RNA in E-Gel Agarose Gels containing SYBR Safe stain. The Safe Imager 2.0 Blue-Light Transilluminator is an independent system designed for viewing stained gels on the benchtop.

Will ethanol precipitation remove the SYBR Safe dye from my DNA?

Yes, SYBR Safe stain is easily removed from nucleic acids by ethanol precipitation or by the ethanol wash steps used for purification spin columns.

Is SYBR Safe stain available in any precast gels?

Several E-Gel products are available with SYBR Safe DNA gel stain. These gels can be used in the same manner as their ethidium bromide counterparts, with the additional safety and application benefits of SYBR Safe. To learn more about these products, search "E-Gel Precast Agarose Gels" from the Thermo Fisher Scientific website home page.

Does use of SYBR Safe stain affect cloning efficiencies after gel purification?

We have found a distinct advantage to using SYBR Safe stain rather than ethidium bromide when purifying DNA from gels for downstream use. SYBR Safe stain is compatible with blue light imaging systems as well as UV. Using blue light to visualize the DNA allows you to purify a band with virtually no UV-induced nicking or crosslinking. This can dramatically increase cloning efficiency. Data from one such experiment showing higher cloning efficiency with PCR products visualized with SYBR Safe and blue light vs. ethidium bromide and UV light can be seen on the information page for Safe Imager 2.0 Blue-Light Transilluminator (Cat. No. G6600) and on the SYBR Safe home page.

Does SYBR Safe stain need to be used in a dark room?

We recommend that SYBR Safe 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.

What is the lower limit of detection of SYBR Safe stain?

SYBR Safe 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.

Is SYBR Safe stain compatible with the same applications as ethidium bromide?

SYBR Safe DNA gel stain is compatible with all downstream applications we have tested so far, including excising PCR products from gels, gel purification, Gateway cloning, TOPO cloning, and restriction enzyme cloning. The use of SYBR Safe DNA gel stain with non-UV blue light emitted by the Safe Imager instrument allows you to purify DNA with virtually no UV-induced nicking or crosslinking compared to ethidium bromide and UV, resulting in dramatically increased cloning efficiencies. If you have a unique application that works with SYBR Safe DNA gel stain, send us the details at techsupport@thermofisher.com.

Why do I sometimes see "speckles" in my gel when using SYBR Safe stain?

Many whitening agents used in clothing, some synthetic fibers, as well as some fungi and bacteria, fluoresce at the same wavelength as SYBR Safe stain. These contaminants within or on the surface of the gel may produce this “speckling”. This can be avoided by being careful with preparation of the gel (i.e. try to keep the gel dust free). Alternatively, to obtain a publication quality image you may be able to preferentially photobleach some contaminants by leaving the gel on the UV box for 15-30 minutes, where the speckles will disappear before the SYBR stain photobleaches.

What are the optimal excitation wavelengths for visualizing SYBR Safe 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 2.0 Blue-Light Transilluminator, Cat. No. G6600). Maximal excitation occurs at 502 nm; the Safe Imager 2.0 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.

Can I use the SYBR Safe stain with my ethidium bromide filter and camera settings?

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.

What are the recommended photographic settings for my gel documentation system when using SYBR Safe stain?

Stained gels can be photographed using Polaroid 667 black-and-white print film and SYBR Safe photographic filter (Cat. No. S37100). Invitrogen SYPRO photographic filter (Cat. No. S6656) or a Kodak Wratten #9 filter also work well. Table 5 in the SYBR Safe product manual lists a filter selection guide for different instruments.

What is the recommended filter for my gel documentation system when using SYBR Safe stain?

Please see the SYBR Safe home page for a list of recommended filters and settings for several different gel documentation instruments. You can find it by searching "SYBR Safe DNA Gel Stain" from the Thermo Fisher Scientific website home page.

If your system is not listed, please contact the instrument manufacturer for a recommendation. Note that the excitation and emission spectra of SYBR Safe gel stain are very similar to those of SYBR Green I, SYBR Green II, and SYBR Gold gel stains, as well as fluorescein (FITC). Therefore, filters appropriate for these dyes can also be used.

How do I use the SYBR Safe photographic filter?

The SYBR Safe photographic filter (Cat. No. S37100) is a Wratten #9 gelatin filter. This filter is a 75 mm x 75 mm sheet of plastic that should be mounted in front of the lens of the camera. With a Polaroid camera and B&W film (#667), the filter may be taped inside the hood or mounted in a cassette and snapped in place inside the hood (the opening in which the camera lens is mounted upon).

For other camera systems, this sheet may be mounted in a cassette or filter-housing and placed in front of the camera lens. An alternative is to use thread-on glass filters of the same rating available from most camera supply vendors. Please note - not all camera systems require use of the SYBR Safe filter. See the SYBR Safe home page for a list of recommended filters and settings for several different instruments - you can find the page by searching "SYBR Safe DNA Gel Stain" from theThermo Fisher Scientific website home page.

Can DNA bands stained with SYBR Safe be seen on a UV transilluminator without the use of emission filters?

DNA bands stained with SYBR Safe stain may be visible by eye on a 300 nm transilluminator if there is a sufficient amount of DNA per band. However, optimal detection is obtained by photographing the gel using a Wratten #9 emission filter (or other filter with a similar rating) with your CCD or film camera. With UV transilluminators (light boxes), UV bulbs may also emit some infrared (IR) wavelengths; if your camera lens is not specially coated to block IR, an IR-blocking filter is needed to prevent the appearance of the UV bulbs under your gel.

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