Lonza Rockland, Inc.
www.lonza.com
biotechserv@lonza.com
Tech Service: 800-521-0390
Customer Service: 800-638-8174
Document # 18110-0807-06
Rockland, ME 04841 USA

Instructions for using SYBR® Green II Nucleic Acid Gel Stain

Introduction
SYBR® Green II Nucleic Acid Gel Stain is one of the mostsensitive dyes known for detecting RNA in electrophoretic gels.
SYBR® Green II Nucleic Acid Gel Stain can be detected as
little as 100 pg of single-stranded DNA per band in agarose or
polyacrylamide gels using 254 nm epi-illumination, black andwhite Polaroid® 667 Print Film and a SYBR® Green Gel Stain
Photographic Filter (Catalog No. 50530) (1,2). Even with 300
nm transillumination, as little as 500 pg RNA per band can bedetected. Our SYBR® Green II Nucleic Acid Gel Stain issignificantly more sensitive than ethidium bromide, the mostcommonly used stain for detecting nucleic acids in gels. With
300 nm transillumination and photography through an orangered gelatin filter, ethidium bromide’s sensitivity limit in astandard agarose minigel is about 1.5 ng single-stranded
nucleic acid per band.

In addition, staining agarose-formaldehyde gels with SYBR®
Green II Nucleic Acid Gel Stain does not interfere with transfer
of RNA to filters or subsequent hybridization in Northern blot
analysis.
SYBR® Green II Nucleic Acid Gel Stain not only should
facilitate the detection of viroid RNAs and multicopy cellular
RNA species, but may also prove useful in applications, suchas single-strand conformation polymorphism (SSCP) analysis(4), that require extremely sensitive detection techniques.
Conventional SSCP analysis requires radioactive hybridization
probes. Although nonisotopic techniques for SSCP analysis
have been developed (5-8) ,they require long complex
procedures, such as silver staining or chemiluminescencemediated signal amplification. Ethidium bromide was used todevelop a method for nonisotopic SSCP in precast
polyacrylamide minigels (9). Not only is this nonisotopic SSCP
technique simple, rapid and reproducible, but it allows precise
temperature control - an important parameter in SSCP
analysis. SYBR® Green II Nucleic Acid Gel Stain should
prove more sensitive than ethidium bromide in suchapplications.

With denaturing agarose-formaldehyde gels andpolyacrylamide-urea gels, the sensitivity of SYBR® Green II
Nucleic Acid Gel Stain is somewhat reduced, though still
superior to that of ethidium bromide. To achieve maximal
sensitivity with ethidium bromide, agarose-formaldehyde gelsmust be washed for several hours prior to staining. In contrast,
without any washing or destaining steps, we have been able todetect 1.0 ng/band RNA in a SYBR® Green II Stained
agarose-formaldehyde gel or polyacrylamide-urea gel using
254 nm epi-illumination, and about 4.0 ng RNA per band using
300 nm transillumination. This remarkable sensitivity of
SYBR® Green II Nucleic Acid Gel Stain for detecting RNA canbe attributed to several factors, including superior fluorescencequantum yield, binding affinity, and fluorescence enhancement.
Although it is not selective for RNA staining, this dye exhibits ahigher quantum yield when bound to RNA (∼0.54) than to
double-stranded DNA (∼0.36). This property is somewhat
unusual among nucleic acid stains; most show far greater
quantum yields and fluorescence enhancements when boundto double-stranded nucleic acids. The fluorescence quantum
yield of the RNA/SYBR® Green II Stain complex is more thanseven times greater than that of the RNA/ethidium bromide(3)complex (∼0.07) . The affinity of SYBR® Green II Nucleic
Acid Gel Stain for RNA is also higher than that of ethidium
bromide, and its fluorescence enhancement upon binding RNA
is well over an order of magnitude greater. Because SYBR®
Green II Nucleic Acid Gel Stain has a low intrinsic
fluorescence, there is no need to destain gels to remove freedye. The fluorescence of RNA/SYBR® Green II Stain
complexes is not quenched in the presence of urea orformaldehyde, eliminating the need to wash these denaturants
out of gels prior to staining.

Storage and Handling
Storage
SYBR® Green II Nucleic Acid Gel Stain is provided either as a
2 x 500 μl dye solution in high-quality, anhydrous DMSO
(Catalog No. 50522) or as a set of 10 vials, each containing 50
μl of DMSO stock solution (Catalog No. 50523). One ml of theconcentrated reagent will prepare a total of 10 liters of working
stain solution, which is sufficient to stain more than 100
agarose or polyacrylamide minigels. When stored frozen at 20°C, protected from light, and dessiccated this stock solution
is stable for six months to one year. We recommend dividing
the stock solution of dye into smaller aliquots that will thaw
faster e.g. 50 μl/vial). Staining reagent diluted in buffer can be
stored protected from light either at 4°C for several weeks or atroom temperature for three or four days. Staining solutions
prepared in water are less stable than those prepared in buffer
and must be used within 24 hours to ensure maximal staining
sensitivity. In addition, staining solutions prepared in bufferswith pH below 7.5 or above 8.0 are less stable and show
reduced staining efficacy. We recommend storing aqueous
stain solutions in plastic rather than glass, as the stain may
absorb to glass surfaces.

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Handling and Disposal
No data is available addressing the mutagenicity or toxicity of®
SYBR® Green II Nucleic Acid Gel Stain. Because thisreagent binds to nucleic acids, it should be treated as apotential mutagen and used with appropriate care. The DMSO
stock solution should be handled with particular caution as
DMSO is known to facilitate the entry of organic molecules into
tissues. We strongly recommend using double gloves whenhandling the DMSO stock solution. As with all nucleic acidstains, solutions of SYBR® Green II Nucleic Acid Gel Stain
should be poured through activated charcoal before disposal.
The charcoal must then be incinerated to destroy the dye.
Refer to the MSDS for safety precautions.

SYBR® Green II Nucleic Acid Gel Stain does not appear tointerfere with enzymatic reactions.
We recommend the addition of 0.1% to 0.3% SDS in the
prehybridization and hybridization solutions when performing
Northern or Southern blots on gels stained with SYBR® Green
II Nucleic Acid Gel Stain.
Nucleic acid bound SYBR® Green II Nucleic Acid Gel Stain
fluoresces green under UV transillumination.
We do not recommend photographing gels with a
254 nm transilluminator. An outline of the UV light source canappear in photographs. A filter that will allow a 525 nm
transmission and exclude other wavelengths (e.g., those in theinfrared) is required.

Spectral Characteristics
Fluorescence Spectra
SYBR® Green II Nucleic Acid Gel Stain may be used with
commonly available ultraviolet epi- and transilluminator
excitation sources, blue-light transilluminators such as theClare Chemical Dark Reader® transilluminator, as well as
hand held ultraviolet lamps. SYBR® Green II Nucleic Acid Gel
Stain is maximally excited at 497 nm, but also has a secondary
excitation peak centered near 254 nm. The fluorescenceemission of SYBR® Green II stained RNA is centered at 520
nm. These spectral characteristics make SYBR® Green II
Nucleic Acid Gel Stain compatible with a wide variety of gel
reading instruments, ranging from those with ultraviolet epiand transillumination to argon laser and mercury-arc lamp
excitation gel scanners.

SYBR® Green II Nucleic Acid Gel Stain is not compatible with
GelBond® Flim or GelBond® PAG Film.
Experimental Protocols
Staining Following Electrophoresis
Perform electrophoresis on nondenaturing gels or on
denaturing polyacrylamide-urea or agarose-formaldehyde gelsaccording to standard techniques (10). SYBR® Green II Nucleic
Acid Gel Stain has not been tested with other gel matrices.

1. Allow each vial to warm to room temperature and thenSYBR® Green Gel Stain Photographic Filter
For optimal sensitivity with black and white film, SYBR® Green
II Stained gels should be photographed through Lonza’s
SYBR®® Green Gel Stain Photographic Filter (Catalog No.
50530). A number of other yellow or green gelatin orcellophane filters (available from Kodak® through photographyequipment suppliers) can also be used for photography, but
most will provide slightly reduced sensitivity. The orange-red
filters used to photograph ethidium bromide-stained gelsshould not be used with SYBR® Green II RNA Stained gels.
The SYBR® Green Gel Stain Photographic Filter should be
stored in a cool, dry place and handled only at its edges. If thefilter needs to be cut to fit into a specialized holder, werecommend first placing it between two sheets of clean, fairly
stiff paper and then cutting out the desired shape with scissors.

briefly centrifuged in a microfuge to deposit the DMSO
solution at the bottom of the vial.

2. Dilute the stock SYBR® Green II Nucleic Acid Gel Stain.
For nondenaturing gels and denaturing polyacrylamideurea gels, we recommend a 1:10,000 dilution in TBE (89mM Tris base, 89 mM Boric acid, 1 mM EDTA, pH 8). For
staining agarose-formaldehyde gels, or glyoxaldenatured RNA, use a 1:5,000 dilution in TBE.

3. Place the gel in a staining container, such as the top of apipet-tip box. Do not use glass or non-polypropylene
plastic staining containers. Add enough staining solution
to cover the gel. Protect the staining container from lightby covering it with aluminum foil or placing it in the dark.
There is no need to wash urea or formaldehyde out of gelsprior to staining.

Application Notes
SYBR® Green II Stain can be removed from the nucleic acidby ethanol precipitation. Isopropanol precipitation is somewhat
less effective at removing the dye; butanol extraction,
chloroform extraction and phenol extraction do not remove thedye efficiently.

4. Gently agitate the gel at room temperature. The optimal
staining time is typically 10-40 minutes for polyacrylamidegels and 20-40 minutes for agarose gels. The staining
time may vary depending on the thickness of the gel andthe percentage of agarose or polyacrylamide. No
destaining is required. The staining solution may be stored
in the dark (preferably refrigerated) and reused three tofour times.

Gels previously stained with ethidium bromide cansubsequently be stained with SYBR® Green II Stain following
the standard protocol for post-staining. There will be somedecrease in sensitivity when compared to a gel stained onlywith SYBR® Green II Nucleic Acid Gel Stain.

Protocol for Adding Dye to Loading Buffer
SYBR® Green II Stain can be added directly to the loading
buffer at a final concentration of 1:1,000. First prepare a
1:100 dilution of SYBR® Green II Nucleic Acid Gel Stain in
high-quality anhydrous DMSO. The 1:100 dilution can be
stored in the freezer and reused. Add 1 μl of this dilution to 9
μl -10 μl of your sample before loading.

Do not dilute SYBR® Green II Stain stock solution in glass
containers because SYBR® Green II Nucleic Acid Gel Stain
will bind to glass. Dilute the stock solution in a polypropylene
container.

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Visualizing and Photographing Stained Gels.
Illuminate the stained gel using 300 nm ultraviolet
transillumination, or for greater sensitivity, 254 nm epiillumination.
Photograph the gel with Polaroid® 667 Black and White Print
Film using a SYBR® Green Gel Stain Photographic Filter.
Stained gels have negligible background fluorescence,
allowing long film exposures when detecting small amounts ofRNA. For 300 nm transillumination, typically a 1-2 second
exposure using an f-stop of 4.5 is adequate. For 254 nm epiillumination (especially with a hand held lamp), exposures on
the order of 1-1.5 minutes may be required for maximal
sensitivity.
Warnings and Precautions
These products are manufactured for Lonza Rockland, Inc., by
Molecular Probes, Inc. and are sold for Research Use Only.
Not for use in diagnostic procedures.SYBR® Green II Nucleic
Acid Gel Stain is the subject of patent applications filed by
Molecular Probes, Inc., and is not available for resale or othercommercial purposes without specific permission of Molecular
Probes, Inc. SYBR® is a trademark of Molecular Probes, Inc.
References
1. FASEB J. 8, A1266 (1994)
2. Biomedical Products. 19, 68 (1994)
3. Cytometry. 7, 508 (1986)
4. Genomics. 5, 874 (1989)
5. Trends in Genetics. 8, (1992)
6. Nucleic Acids Res. 19, 405 (1991)
7. Nucleic Acids Res. 19, 2500 (1991)
8. Nucleic Acids Res. 19, 3154 (1991)
9. Nucleic Acids Res. 21, 3637 (1993)
10. Molecular Cloning: A Laboratory Manual,
Second Edition, J. Sambrook, E.F. Fritsch and T.
Maniatis, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, New York (1989).
Ordering information
Catalog
No.
50522
50523
50530

Description

Size

SYBR® Green II RNA Gel
Stain
SYBR® Green II RNA Gel
Stain
SYBR® Green Photographic
Filter2 x 500 μl
10 x 50 μl
3“ square

Related Products
Lonza Agarose Products
MDE® Gel Solution
Latitude® Precast Gels
Reliant® Gel System
AccuGENE® MOPS Buffer
GelStar and GelBond are trademarks of FMC Corporation,SYBR is a trademark of Molecular Probes, Inc. Polaroid is atrademark of Polaroid Corp. Kodak is a trademark of Eastman
Kodak Co. All other trademarks herein are marks of the Lonza
Group or its affiliates. SYBR Green Stain is covered by US
Patent 5,436,134 and 5,658,751. Other US and foreign
patents pending.

For more information contact Technical Service at (800)521-0390 or visit our website at www.Lonza.com.

© 2007 Lonza Rockland, Inc.
All rights rights reserved.

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