Less hazardous, sensitive DNA gel stain

Invitrogen™ SYBR™ Safe DNA Gel Stain is a highly sensitive stain for visualization of DNA in agarose or acrylamide gels. SYBR Safe stain is specifically formulated to be a less hazardous alternative to ethidium bromide that can be used with either blue-light or UV excitation.

SYBR Safe stain is supplied as either a concentrate or a ready-to-use solution that can be used like an ethidium bromide solution. The stain is also suitable for staining RNA in gels.

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More colonies from your cloning

Our scientists have demonstrated a vast improvement in cloning efficiency of DNA fragments stained with SYBR Safe stain and visualized using the Invitrogen™ Safe Imager™ Blue-Light Transilluminator versus the same fragments stained with ethidium bromide and exposed to UV light. 

Get enhanced restriction enzyme cloning efficiency

The Invitrogen™ pCMV•SPORT-βgal plasmid vector was double digested with EcoRI and HindIII to create two sticky-ended DNA fragments: the lacZ gene (3,536 bp) and the backbone of the vector (4,318 bp). Equal amounts of digested DNA were electrophoresed on 1% agarose gels. The gels were stained with either ethidium bromide (0.5 mg/mL in TBE) or SYBR Safe DNA gel stain (1:10,000 dilution in TBE) for 15 minutes, and then viewed using either a UV tranilluminator or the Safe Imager™ blue-light transilluminator, respectively. After defined exposure times, the two DNA fragments were excised from the gels and purified using the Invitrogen™ PureLink™ Gel Extraction Kit. Control DNA fragments were stained but not exposed to transillumination. The lacZ gene and the vector backbone were religated using Invitrogen™ T4 DNA Ligase chemically transformed into Invitrogen™ UltraMax™ DH5α™-FT™ cells, and plated onto selection plates. The total number of blue and white colonies was counted to evaluate cloning efficiency. Each experiment was conducted in triplicate, and the average cloning efficiency was determined.

The total number of colonies on the ethidium bromide/UV plates was significantly lower than that on SYBR Safe/Safe Imager plates even at the earliest time points (Figure 1). Over the course of the various time points taken, the number of viable colonies in the ethidium bromide/UV set of plates, dropped from roughly 15,000 colonies to less than 500 colonies, representing an approximately 30-fold reduction in cloning efficiency. In contrast, plates in the SYBR Safe/Safe Imager set maintained a near control level of colonies (>12,500) at all time points, including the 120-second time point.

Enhanced restriction enzyme cloning efficiency
Figure 1. Enhanced restriction enzyme cloning efficiency. The lacZ insert fragment and pCMV•SPORT-βgal vector backbone were electrophoresed on duplicate agarose gels and stained with either SYBR Safe DNA gel stain or ethidium bromide. The DNA fragments were then visualized for defined periods using either the Safe Imager blue-light transilluminator or a UV transilluminator, respectively. Insert and vector DNA bands were excised from the gels, ligated, chemically transformed into competent bacteria and plated on ampicillin–X-gal plates (representative plates shown in panel A). Cloning efficiency was determined based on the total number of blue and white colonies present (B). Data plotted reflect an average of three experiments.

Benefit from improved Gateway cloning efficiency

In the experiment, a 1.25 kb gene was amplified by PCR. Seven equal amounts of the PCR product were electrophoresed on duplicate agarose gels; one gel was visualized with SYBR Safe DNA gel stain and blue-light transillumination, while the other gel was visualized with ethidium bromide and UV transillumination. Bands were excised after defined exposure times; the products purified using the PureLink Gel Extraction Kit, then used in an Invitrogen™ Gateway™ BxP cloning reaction. A portion of each reaction was transformed into Invitrogen™ One Shot™ TOP10 chemically competent bacteria; and three serial dilutions were plated. The results showed an 80% reduction in colony number after only 30 seconds exposure to ethidium bromide/UV (Figure 2). After only 2 minutes exposure, the colony count was nearly zero. In contrast, the cloning efficiency attained using SYBR Safe DNA gel stain and Safe Imager blue-light transillumination remained at virtually 100% of the control value throughout the entire time course of the experiment.

Figure 2. Improved Gateway cloning efficiency. A 1.25 kb gene was amplified by PCR using attB1 and attB2 primers, and equivalent fractions were separated on duplicate agarose gels. Gels were stained with either SYBR Safe DNA gel stain or ethidium bromide, and DNA fragments were visualized with either the Safe Imager blue-light transilluminator or a UV transilluminator, respectively. The PCR products were cloned by Gateway BP recombination and transformed into chemically competent bacteria. Three serial dilutions were plated, and colonies were counted.

Conclusion

The use of SYBR Safe DNA gel stain and Safe Imager blue-light transilluminator significantly improves cloning efficiency in both restriction enzyme as well as Gateway cloning methods.

 

Safe Imager Blue-Light Transilluminator

Eliminate the safety concerns of UV transillumination

  • Does not damage your skin and eyes
  • Produces brighter light and more uniform emission than conventional blue-light sources
  • Provides optimal excitation for SYBR Safe DNA gel stain
  • Optimized for use with other Invitrogen™ nucleic acid and protein stains such SYBR™ Gold, SYBR™ Green I & II, SYPRO™ Ruby, SYPRO™ Orange, and Coomassie Fluor™ Orange stains

Instrument specifications

  • Overall dimensions: 28 × 31 × 7 cm (11 × 12.25 × 2.75 in)
  • Viewing surface dimensions: 20 × 20 cm (7.87 × 7.87 in)
  • Light source: light emitting diodes (LED) producing a narrow emission peak centered at ~470 nm
  • LED life: 100,000 hours
  • Included accessories: amber filter unit, viewing glasses, and international power cord
      

Assessment of mutagenicity and environmental safety: SYBR Safe DNA gel stain

 

Assay for morphological transformation in primary cultures

An independent laboratory investigated the ability of SYBR Safe DNA gel stain for inducing an increase in morphological transformation of Syrian hamster embryo (SHE) cells, relative to vehicle control cultures, following a 7-day exposure period. None of the three treatment groups (0.0500, 0.150, and 0.300 µg/mL) induced a significant increase in morphological transformation compared to the concurrent vehicle control. In addition, a significant increase of the morphological transformation frequency was also obtained from the positive control treatment with benzo pyrene at 5.0 mg/mL. The test article (SYBR Safe DNA gel stain) was therefore evaluated as negative in the screening SHE cell transformation assay under 7-day exposure conditions of this study.

 

Conclusion—SYBR Safe DNA gel stain does not induce transformations in primary cultures of Syrian hamster embryo (SHE) cells when compared with solvent alone, strongly indicating that the SYBR Safe DNA gel stain is noncarcinogenic. In contrast, ethidium bromide tests positive in the SHE assay, consistent with its known activity as a strong mutagen.

Assays for chromosomal aberations and forward mutations

An independent laboratory investigated the ability of SYBR Safe DNA gel stain to induce chromosomal aberrations in cultured peripheral blood lymphocytes with and without exogenous metabolic activation. No significant increases in the number of cells with structural aberrations, polyploidy, or endoreduplication were observed in test either with or without S9 activation. Similarly, no increases in the mutant frequency were observed that exceeded the minimum criteria in L5178Y TK+/- mouse lymphoma cells.

Conclusion—SYBR Safe DNA gel stain does not cause mutations in mouse lymphoma cells at the TK locus, nor does it induce chromosomal aberrations in cultured human peripheral blood lymphocytes, with or without S9 metabolic activation, using standardized tests against appropriate controls.

Summarized data: mammalian genotoxicity analysis of SYBR Safe DNA gel stain

In vitro test* Cell type Result with S9 activation† Result without S9 activation†
Transformation1 Syrian hamster embryo (SHE) cells NA Negative
Chromosomal aberrations2 Cultured human peripheral blood lymphocytes Negative Negative
Forward mutation3,4 L5178YTK+/- mouse lymphoma cells Negative Negative

* In vitro tests were performed by Covance Laboratories Inc., Vienna, VA. † Mammalian S9 fraction obtained from Aroclor 1254 induced rat liver. NA = Not applicable. 1Fundamental and Molecular Mechanisms of Mutagenesis 356, 1 (1996); 2Evans, H.J., in Chemical Mutagens, Principles and Methods for Their Detection, A. Hollaender, Ed., Vol. 4, (1976) pp. 129; 3Mutation Res 72, 447 (1980); 4Mutation Res 59, 61 (1979).

Assay for reverse mutations (Ames test)

Samples were pretreated with a mammalian S9 fraction and then tested. With S. typhimurium strains TA97a, TA98, TA100 and TA102, an increase in revertants of more than twofold over background indicates a positive result for mutagenicity in this test. With strains TA1535, TA1537 and TA1538, an increase in revertants of more than threefold over background indicates a positive result.

Conclusion—Compared to ethidium bromide, SYBR Safe DNA gel stain causes fewer mutations in the standard Ames test, as measured in several different strains of Salmonella typhimurium. Weakly positive results for SYBRSafe DNA gel stain in this test occurred in four out of seven strains and only with activation by a mammalian S9 fraction (see figure).








Assay for oral toxicity

A single oral administration of SYBR Safe DNA gel stain in 0.5X TBE at a limit dose of 5,000 mg/kg to three female rats produced no mortalities or toxic signs. The procedure is designed to determine that acute oral toxicity of the material under test. A Limit Screen test was performed using three female Sprague Dawley rats, which received an oral limit dose of 5,000 mg/kg of SYBR Safe DNA gel stain. The animals were observed for mortality, weight change, and toxic signs for a two-week period. Since all three rats survived for two weeks after the dose administration, the LD50 for the test article was considered to be greater than the Limit Dose and no additional testing was required.

Conclusion—A single oral administration of SYBR Safe DNA gel stain produces no signs of mortality or toxicity at a limit dose of 5,000 mg/kg.

Summarized data: oral toxicity

Analysis Method Results
Aquatic toxicity* Fathead minnow CA Title 22 acute screening Not hazardous or toxic to aquatic life
Oral toxicity** EPA Acute Oral Toxicity Test OPPTS 870.1100 LD50 > 5000 mg/kg

* Performed by AMEC Earth and Environmental San Diego Bioassay Laboratory, San Diego, CA.
** Performed by Northview Pacific Laboratories, Inc., Hercules, CA.

 

    

Environmental impact results (USA)

Based on extensive environmental safety testing, SYBR Safe DNA gel stain is not classified as hazardous waste under U.S. Federal regulations (Resource Conservation and Recovery Act (RCRA)). SYBR Safe DNA gel stain meets the requirements of the Clean Water Act and the National Pollutant Discharge Elimination System (NPDES) requirements.

Conclusions

  • SYBR Safe DNA gel stain is not classified as corrosive, ignitable, or reactive under the guidelines of the Environmental Protection Agency (EPA) (Table 1).
  • 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 (Table 2).
  • 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 (Table 3). 

 

Table 1. SYBR Safe DNA gel stain hazardous waste analysis.

Analysis* Method Results
Corrosivity EPA 150.1 Not corrosive (pH = 8.25)
Corrosivity (by Corrositex) DOT-E 10904 Category 2 noncorrosive
Ignitability EPA 1010 Not ignitable (<212°F)
Reactivity EPA 9010B/9030A No reactivity detected

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

 

Table 2. SYBR Safe DNA gel stain: overview of NPDES analysis*.

Test SYBR Safe stain in 0.5X TBE 0.5X TBE
PH (150.1) 8.45 8.48
Total cyanide (335.2) None detected None detected
Chemical oxygen demand (COD; 410.1) 7020 6840
Ammonia as nitrogen (350.1) 253 248
Total organic carbon (415.1) 2480 2360
Total phenoloics (420.1) None detected None detected
Organochlorine pesticides and PCBs (608M) None detected None detected
Semi-volatile organic compounds (625) None detected None detected
Volatile organic compounds (624) None detected None detected
Priority pollutant metals (Sb, As, Be, Cd, Cr, Cu, Pb, Hg, Ni, Se, Ag, Tl, Zn) (EPA 200.7/200 series) None detected None detected

* 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.

 

Table 3. SYBR Safe DNA gel stain: detailed organic compound analysis*.

Organochlorine pesticides and PCBs (608M)
alpha-BHC beta-BHC gamma-BHC delta-BHC (Lindane) Heptachlor
Aldrin Heptachlor Epoxide Endosulfan I Dieldrin 4,4'-DDE
Endrin Endosulfan II 4,4'-DDD Endrin Aldehyde Endosulfan Sulfate
4,4'-DDT Toxaphene Chlordane Aroclor 1016 Aroclor 1221
Aroclor 1232 Aroclor 1242 Aroclor 1248 Aroclor 1254 Aroclor 1260
Ethylbenzene Bromoform 1,1,2,2,-Tetrachloro
ethane		 1,3-Dichloro
benzene		  
Volatile organic compounds (624)
Chloromethane Vinyl Chloride Bromomethane Chloroethane Trichlorofluoro
methane
1,1-Dichloroethene Methylene Chloride trans-1,2-Dichloroethene 1,1-Dichloroethane Chloroform
1,1,1-Trichloroethane (TCA) Carbon Tetrachloride Benzene 1,2-Dichloroethane (EDC) Trichloroethene (TCE)
1,2-Dichloropropane Bromodichloro
methane		 2-Chloroethyl Vinyl Ether trans-1,3-Dichloro-
propene		 Toluene
cis-1,3-Dichloro
propene		 1,1,2-Trichloroethane Tetrachloroethene (PCE) Dibromochloro
methane		 Chlorobenzene
1,4-Dichlorobenzene 1,2-Dichlorobenzene Acrolein Acrylonitrile  
Semi-volatile organic compounds (625)
N-Nitrosodimethyl-
amine		 Bis(2-chloroethyl) Ether Phenol 2-Chlorophenol Bis (2-chloroisopropyl) Ether
Hexachloroethane N-Nitrosodi-n-propyl-
amine		 Nitrobenzene Isophorone 2-Nitrophenol
2,4-Dimethylphenol 4-Nitrophenol 2,4-Dichlorophenol 1,2,4-Trichlorobenzene Naphthalene
Hexachlorobutadiene 4-Chloro-3-methylphenol Hexachlorocyclo-
pentadiene		 2,4,6-Trichlorophenol 2-Chloronap
hthalene
Acenaphthylene Dimethyl Phthalate 2,6-Dinitrotoluene Acenaphthene 2,4-Dinitrophenol
Bis (2-chloroethoxy) methane 2,4-Dinitrotoluene Fluorene Diethyl Phthalate 2-Methyl-4,6-dinitrophenol
N-Nitrosodiphenyl
amine		 4-Bromophenyl Phenyl Ether Hexachlorobenzene Pentachlorophenol Phenanthrene
Anthracene Di-n-butyl Phthalate Fluoranthene Benzidine Pyrene
Butyl Benzyl Phthalate 3,3'-Dichlorobenzidine Benz(a)anthracene Chrysene Bis(2-ethylhexyl) Phthalate
Di-n-octyl Phthalate Benzo(b)fluoranthene Benzo(k)fluoranthene Benzo(a)pyrene Indeno(1,2,3-cd)pyrene
Dibenz(a,h)anthracene Benzo(g,h,I)perylene 1,2-Diphenylhydrazine 2,3,7,8-Tetrachloro-
dibenzo-p-dioxin		 4-Chlorophenyl Phenyl Ether

* 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.

Bound to nucleic acids, SYBR Safe stain has fluorescence excitation maxima at 280 and 502 nm, and an emission maximum at 530 nm. Stained DNA can be visualized and analyzed using imaging systems equipped with an excitation source in the UV range or between 470 nm and 530 nm.

Filter recommendations for use With SYBR Safe DNA gel stain

The table below 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 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.
 

Instrument Manufacturer Excitation source Emission filter
Polaroid® camera Polaroid® UV Invitrogen™ SYBR™ Safe photographic filter (Cat. No. S37100)
FCR-10 Polaroid® UV #3-4218
AlphaImager Alpha Innotech 302 nm SYB-500
AlphaDigiDoc RT Alpha Innotech UV transilluminator  
Shroud, camera stand Alpha Innotech UV transilluminator  
DE500 or DE400 light cabinet 2.17" diam. Alpha Innotech UV transilluminator SYB-100
DE500 or DE400 light cabinet 2" diam. Alpha Innotech UV transilluminator SYB-500
VersaDoc Imaging Systems Bio-Rad Broadband UV 520LP
Molecular Imager FX Systems Bio-Rad 488 nm 530 DF 30
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 (fluorescence mode)  
ImageMaster 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
FOTO/Analyst Express/Investigator/Plus/Luminary Fotodyne UV Fluorescent Green (#60-2034)†
FOTO/Analyst Express/Investigator/Plus/Luminary Fotodyne UV Fluorescent Green (#62-4289)†
FOTO/Analyst Minivisionary Fotodyne UV Fluorescent Green (#62-2535)†
FOTO/Analyst Apprentice Fotodyne UV Fluorescent Green (#60-2056)†
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 535 535 nm WB50
Mini BIS/Mini BIS Pro DNR Bioimaging UV 320 Yellow
Compact BIS DNR Bioimaging UV 320 Orange
Syngene Instruments Syngene UV 500–600 nm shortpass filter

†Optional filters available from respective manufacturer

Case Study 1: Forsyth Institute, Boston, MA, USA.

  The following data was provided by the EHS office at the Forsyth Institute. They calculated the total institutional costs of using SYBR Safe DNA gel stain versus ethidium bromide for staining DNA gels. By taking into account the stain disposal costs of ethidium bromide, they determined that using SYBR Safe DNA gel stain resulted in a 14% cost savings for their institution.
  SYBR Safe Ethidium bromide
Reagent costs 400 µl concentrate @ $40 per bottle
5 µl concentrate per gel
$0.50 per gel		 10 mL of 10 mg/mL concentrate @ $32 per bottle
5 µL concentrate per gel
$0.016 per gel
Disposal costs - materials
$0.00		 20 L capacity charcoal filter @ $33 per filter
0.1 L per gel
$0.165 per gel
Disposal costs -time & labor $0.00 4 hours labor @ $20 per hour to filter 20 L of waste
0.1 L of waste per gel
$0.40 per gel
Overhead disposal costs $0.00 Water usage (suction for filtration)
Hazardous waste fees (for filter disposal)
Additional uncalculated costs
Total cost per gel $0.50 $0.58++

Note:  this data was derived from a specific institution; actual costs at other institutions may vary.

Benefits of SYBR Safe DNA gel stain

  • 14% cost savings
  • Technician labor savings
  • Safer working environment
  • Environmental benefits
  • No hazardous waste
  • No wasted water

 

Case Study 2: Iowa State University, Ames, IA, USA.

 

ISU pours SYBR Safe DNA gel stain down the drain

Wastewater officials in Ames, Iowa, United States, have approved the disposal of SYBR Safe DNA gel stain as nontoxic waste, allowing the diluted dye solution to be poured directly down the drain at Iowa State University (ISU).

The EHS office at ISU, presented the safety data for SYBR Safe DNA gel stain to the Ames Wastewater Treatment Facility. The data included results from US National Pollutant Discharge Elimination System tests, which showed that 0.5X TBE containing SYBR Safe DNA gel stain demonstrated no significant increase in discharges over 0.5X TBE alone. Following an independent review by wastewater engineers, city officials gave the green light for ISU to treat SYBR Safe DNA gel stain as nontoxic waste.

For Research Use Only. Not for use in diagnostic procedures.