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.
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.
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.
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.
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 drainWastewater 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). |
|
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 drainWastewater 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). |
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