SYBR® Safe DNA Gel Stain Frequently Asked Questions
Get quick answers to most of your questions. Frequently asked questions have been grouped into three categories.
How should I dispose of SYBR® Safe DNA gel stain?
Some institutions and municipalities have approved the disposal of 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.
Is SYBR® Safe DNA gel stain really safe? Do I have to use gloves when I use it?
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.
Is SYBR® Safe DNA gel stain the same as SYBR® Green I dye?
All 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. SYBR® Green I is an ultrasensitive stain for dsDNA, and SYBR® Green II is a highly sensitive stain for RNA and ssDNA. All SYBR® dyes are optimally excited by the Safe Imager™ blue-light transilluminator.
Can agarose gels be cast with SYBR® Safe DNA gel stain in them?
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.
How many parts per million (ppm) of SYBR® Safe DNA gel stain are there in a 1X solution of the stain?
The SYBR® Safe DNA gel stain content in a 1X solution is less than 1 ppm.
How should SYBR® Safe DNA gel stain spills be cleaned up?
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.
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 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.
What are the advantages of using blue light to view DNA stained with SYBR® Safe DNA gel stain?
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.
Where can I find more safety information about SYBR® Safe DNA gel stain?
Our website (www.lifetech.com/sybrsafe) contains all of our latest testing results.
Can bands be seen on a UV transilluminator without the use of emission filters?
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.
How do I use the SYBR® Safe photographic filter?
The 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.
What is the recommended filter for my gel documentation system?
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 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.
What are the recommended photographic settings for my gel documentation system?
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.
Can I view SYBR® Safe DNA gel 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 other excitation wavelengths can I use to visualize 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 (Cat. No. G6600). 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.
Why do I sometimes see “speckles” in my gel when using SYBR® Safe DNA gel 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.”
Is SYBR® Safe DNA gel 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. If you have a unique application that works with SYBR® Safe DNA gel stain, send us the details at firstname.lastname@example.org.
How much of the 10,000X concentrate should I use?
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.
What is the lower limit of detection of SYBR® Safe DNA gel stain?
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.
Can I reuse SYBR® Safe DNA gel stain for a second gel?
We strongly discourage the reuse of SYBR® Safe DNA gel stain, as this practice significantly lowers sensitivity.
Is SYBR® Safe DNA gel stain microwaveable?
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.
Does SYBR® Safe stain DNA gel need to be used in a darkroom?
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.
Does SYBR® Safe DNA gel stain affect cloning efficiencies?
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 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.
Is SYBR® Safe DNA gel stain available in any precast gels?
Several 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 E-Gel® precast gels with SYBR® Safe DNA gel stain, visit www.lifetech.com/egels.
Does ethanol precipitation remove the dye?
SYBR® Safe DNA gel stain is easily removed from nucleic acids by ethanol precipitation.
Which direction does the dye run during electrophoresis?
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 (voltage, wattage, length of run, etc).This effect can be partially counteracted by adding SYBR® Safe DNA gel stain to the running buffer.