Using SYBR Safe™ DNA Gel Stain - Invitrogen

Filter recommendations
for use with SYBR Safe DNA gel stain
Instrument (Manufacturer) Excitation Source Emission Filter
AlphaImager (Alpha Innotech) 302 nm SYB-500
AlphaImager HP (Alpha Innotech) 302 nm SYB-500
AlphaDigiDoc RT (Alpha Innotech) UV transilluminator Not applicable
Shroud, Camera Stand (Alpha Innotech) UV transilluminator SYB-100
DE500 or DE400 light cabinet 2.17” diam. (Alpha Innotech) UV transilluminator SYB-500
DE500 or DE400 light cabinet 2” diam. (Alpha Innotech) UV transilluminator SYB-400
VersaDoc Imaging Systems (Bio-Rad) Broadband UV 520LP
Molecular Imager FX Systems (Bio-Rad) 488 nm 530 nm BP
Gel Doc Systems (Bio-Rad) 302 nm 520DF30 (#170-8074)
Typhoon 9400/9410 (GE Healthcare) 488 nm 520 BP 40
Typhoon 9200/9210/8600/8610 (GE Healthcare) 488 nm 526 SP
FluorImager (GE Healthcare) 488 nm 530 DF 30
Storm (GE Healthcare) Blue (fl uorescence mode) Not applicable
VDS-CL (GE Healthcare) Transmission UV Low
Ultracam/Gel Imager (Ultra-Lum) UV Yellow Filter (#990-0804-07)
Omega Systems (Ultra-Lum) UV 520 nm
Polaroid Camera (Polaroid) UV SYBR Safe Photographic Filter (S37100)
FOTO/Analyst Express/Investigator/Plus/Luminary
(Fotodyne)
UV Fluorescent Green (#60-2034)
FOTO/Analyst Minivisionary (Fotodyne) UV Fluorescent Green (#62-4289)
FOTO/Analyst Apprentice (Fotodyne) UV Fluorescent Green (#62-2535)
FOTO/Analyst Luminary (Fotodyne) UV Fluorescent Green (#60-2056)
FCR-10 (Polaroid) UV #3-4218
FUJI FLA-3000 (FUJI Film) 473 nm 520LP
BioDocIt/AC1/EC3/BioSpectrum (UVP) 302 nm SYBR® Green (#38-0219-01) or SYBR® Gold (#38-0221-01)
Gel Logic (Kodak) UV 535 nm WB50
Syngene Instruments (Syngene) UV 500–600 nm Shortpass fi lter
SYBR Safe DNA Gel Stain
62923 TC562-1
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Using SYBR Safe DNA Gel Stain
SYBR Safe DNA Gel Stain

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Using SYBR Safe DNA Gel Stain
You have questions, we have answers.
SYBR Safe DNA gel stain was designed to replace ethidium bromide—a known mutagen—while offering uncompromised
staining performance. SYBR Safe DNA gel stain is less mutagenic, less toxic, more environmentally friendly, and it works with
traditional gel staining methods.
The practical information contained in this brochure represents many hours of bench experience with SYBR Safe stain.
We hope these guidelines will help you easily and effectively bring SYBR Safe stain into your lab. If you’re ready to put the
potential health risks of ethidium bromide in your past, SYBR Safe DNA gel stain is the answer.
Safety
Q How should I dispose of SYBR Safe stain?
A Some institutions and municipalities have approved the
disposal of SYBR Safe stain directly into their waste
water systems. However, disposal regulations vary—
please contact your safety office or local municipality for
disposal guidelines.
Q Is SYBR Safe stain really safe? Do I have to use gloves
when I use it?
A In numerous tests carried out by independent, licensed
testing laboratories, SYBR Safe stain showed little
or no genotoxicity and no acute toxicity. This stain is
not classified as hazardous waste under U.S. federal
regulations; nevertheless, please exercise common safe
laboratory practice when using this reagent.
Q Where can I find more safety information about
SYBR Safe stain?
A Our website (probes.invitrogen.com/sybrsafe) contains
all of our latest testing results.
Q I work in an institution/city/state/country that requires
additional tests to allow me to dispose of this down the
drain. Will Invitrogen perform these tests?
A Safety and environmental testing of SYBR Safe stain is
ongoing. Please contact us at sybrsafe@invitrogen.com.
We will work with you to obtain disposal approval.
Imaging
Q Can bands be seen on a UV transilluminator without
the use of emission filters?
A Bands stained with SYBR Safe stain are visible to the
eye on a 300 nm transilluminator. However, maximal
detection is obtained by photographing the gel by
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.
Q How do I use the SYBR Safe photographic filter?
A 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.
Q What is the recommended filter for my gel documentation
system?
A The table appearing later in this brochure lists recommended
filters for specific gel documentation systems.
If your system is not listed, contact the manufacturer for
recommendations. Note that the excitation and emission
spectra of SYBR Safe 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.
Q What are the recommended photographic settings for
my gel documentation system?
A 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.
Q Can I use SYBR Safe stain with my ethidium bromide
filter and camera settings?
A Some ethidium bromide filters allow the transmission
of all light above 500 nm. These filters (which often
appear yellow) and their associated camera settings
can be used with SYBR Safe stain, usually with only
minor adjustments to the exposure or gain. Other
ethidium bromide filters (often red in appearance) only
transmit light around or above 600 nm; these filters
and their associated camera settings are not suitable
for use with SYBR Safe stain.
Q What other excitation wavelengths can I use to visualize
SYBR Safe stain?
A SYBR Safe stain has two main excitation peaks: in the
UV 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
Invitrogen’s Safe Imager (coming September 2005) and
Clare Chemical’s Dark Reader). The full excitation and
emission spectra for SYBR Safe stain are provided on
our website (probes.invitrogen.com/sybrsafe) and in
the protocol provided with the stain.
Q Why do I sometimes see “speckles” in my gel when
using SYBR Safe stain?
A Many whitening agents used in clothing, 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.”
Q How can I get a SYBR Safe T-shirt?
A Send us an image of SYBR Safe stain obtained on your
imaging system. If your image is the first received for
that imaging system, we will send you a SYBR Safe
T-shirt. Send images to sybrsafe@invitrogen.com;
remember to include the complete photographic
details of your image and imaging system.
Application
Q Is SYBR Safe stain compatible with the same
applications as ethidium bromide?
A SYBR Safe stain is compatible with all downstream
applications we have tested so far, including excising
PCR products from gels, gel purification, Gateway®
cloning, and restriction enzyme cloning. If you have a
unique application that works with SYBR Safe stain,
send us the details at SYBRSafe@invitrogen.com and
receive a SYBR Safe T-shirt.
Q How much of the 10,000X concentrate should I use?
A Dilute SYBR Safe stain concentrate 10,000-fold in
TAE or TBE buffer prior to use. 50 mL of 1X stain is
sufficient for most minigels (e.g., dilute 5 µL of con-
centrate with 50 mL buffer). For larger gels, increase
volumes proportionally, ensuring that the entire gel
is fully immersed during staining.
Q What is the lower limit of detection of SYBR Safe stain?
A 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.
Q Can I reuse SYBR Safe stain for a second gel?
A We strongly discourage the reuse of SYBR Safe stain,
as this practice significantly lowers sensitivity.
Q Is SYBR Safe stain microwaveable?
A SYBR Safe stain may be briefly microwaved with no
loss of performance. However, we do not know the
effect of repeated or very long duration microwaving.
SYBR Safe DNA Gel Stain
Q Does SYBR Safe stain need to be used in a dark room?
A 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.
Q Does SYBR Safe stain affect cloning efficiencies?
A 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 non-UV blue light
imaging systems—you can therefore purify DNA with
virtually no UV-induced nicking or crosslinking and
achieve dramatically increased cloning efficiency.
Q Is SYBR Safe stain available in any precast gels?
A E-Gel® 1.2% and E-Gel® 2% products are now available
with SYBR Safe stain. These gels can be used in the
same manner as their ethidium bromide counterparts.
For more information on E-Gels® with SYBR Safe, visit
www.invitrogen.com/egels.
Q Does ethanol precipitation remove the dye?
A SYBR Safe stain is easily removed from nucleic acids
by ethanol precipitation.
Q Which direction does the dye run during electrophoresis?
A Similar to ethidium bromide, SYBR Safe stain runs
in the direction opposite migrating DNA. This has no
practical effect on the use of precast gels containing
SYBR Safe stain, as only the very bottom edge of the
gel will have a lower concentration of stain. This effect
can be partially counteracted by adding SYBR Safe
stain to the running buffer.
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