SYBR Safe versus ethidium bromide Nikita Warner Ph

SYBR Safe™ versus ethidium bromide Nikita Warner, Ph. D. Assoc. Product Manager Nucleic Acid Separation & Analysis

What is SYBR Safe™? A DNA gel stain that is: • Better for you – displays no acute oral toxicity and greatly reduced genotocixity • Better for the environment – offers non-hazardous waste status and Clean Water Act compliance • Better for your institution – reduces waste storage and disposal costs • Better for your sample – improves cloning efficiency when used with blue-light illumination without compromising detection sensitivity as compared to ethidium bromide SYBR Safe™ is a better DNA gel stain 2

How is SYBR Safe™ better for me? 3

Oral toxicity evaluation Analysis Method Results Aquatic toxicity* Fathead minnow CA Title Not hazardous or toxic to 22 acute screening aquatic life Oral toxicity** EPA Acute Oral Toxicity Test OPPTS 870. 1100 LD 50 > 5000 mg/kg * Performed by AMEC Earth and Environmental San Diego Bioassay Laboratory, San Diego, CA. ** Peformed by Northview Pacific Laboratories, Inc. , Hercules, CA. Displays no acute oral toxicity 4

Genotoxicity studies In Vitro Test* Cell Type Result with S 9 Activation Result without S 9 Activation Transformation 1 Syrian hamster embryo (SHE) cells NA Negative Chromosomal aberrations 2 Cultured human peripheral Negative blood lymphocytes Negative Forward mutation 3, 4 L 5178 YTK+/- mouse lymphoma cells Negative * In vitro tests were performed by Covance Laboratories Inc. , Vienna, VA. Mammalian S 9 fraction obtained from Aroclor 1254 induced rat liver. NA = Not applicable. 1 Fundamental and Molecular Mechanisms of Mutagenesis 356, 1 (1996); 2 Evans, H. J. , in Chemical Mutagens, Principles and Methods for Their Detection, A. Hollaender, Ed. , Vol. 4, (1976) pp. 129; 3 Mutation Res 72, 447 (1980); 4 Mutation Res 59, 61 (1979). Displays greatly reduced genotocixity 5

Ames test results Causes fewer mutations in the standard Ames test 6

How is SYBR Safe™ better for the environment? 7

Hazardous waste testing According to the guidelines of the EPA, SYBR Safe™ DNA gel stain is not • corrosive • ignitable, or • reactive Analysis* Method Results Corrosivity EPA 150. 1 Not corrosive (p. H = 8. 25) Corrosivity (by Corrositex) DOT-E 10904 Category 2 noncorrosive Ignitability EPA 1010 Not ignitable (<212°F) Reactivity EPA 9010 B/9030 A No reactivity detected * All tests were performed by AMEC Earth and Environmental San Diego Bioassay Laboratory, San Diego, CA. Not classified as hazardous waste under U. S. federal regulations 8

NPDES analysis Tests performed according to National Pollutant Discharge Elimination System (NPDES) guidelines indicate that SYBR Safe™ DNA gel stain does not differ significantly from 0. 5 X TBE buffer Test SYBR Safe Stain in 0. 5 X TBE PH (150. 1) 8. 45 8. 48 Total cynaide (335. 2) 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 Organochlorine pesticides and PCBs (608 M) None detected Semi-volatile organic compounds (625) None detected Volatile organic compounds (624) 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 * 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. SYBR Safe™ DNA gel stain does not differ significantly from 0. 5 X TBE buffer 9

Organic compound analysis Tested were performed on 0. 5 X TBE and 1 X SYBR Safe™ in 0. 5 X TBE for: • Organochlorine pesticides and PCBs (608 M) • Volatile organic compounds (624) • Semi-volatile organic compounds (625) None of the organic pollutant compounds tested for were detected in either sample SYBR Safe™ DNA gel stain in 0. 5 X TBE is indistinguishable from 0. 5 X TBE alone 10

How is SYBR Safe™ better for my institution? 11

SYBR Safe™ Institutional Savings Case Study: Forsyth Institute, Boston, MA (data provided by Forsyth Institute) Ethidium bromide SYBR Safe™ Reagent Costs • 10 ml of 10 mg/ml concentrate @ $32 per bottle • 5 µl concentrate per gel • $0. 016 per gel Disposal costs - Materials • 20 L capacity charcoal filter @ $33 per filter • 0. 1 L per gel • $0. 165 per gel Disposal costs - Time & Labor • 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 ++ Water usage (suction for filtration) Hazardous waste fees (for filter disposal) Additional uncalculated costs Total ethidium bromide cost $0. 58++ per gel Reagent Costs • 400 µl concentrate @ $40 per bottle • 5 µl concentrate per gel • $0. 50 per gel Disposal costs – Materials • $0. 00 Disposal costs - Time & Labor • $0. 00 Overhead disposal costs • None • Total SYBR Safe™ cost • $0. 50 per gel For more details, visit: www. invitrogen. com/sybrsafe 14% institutional savings at a Boston institute Invitrogen Proprietary and Confidential

SYBR Safe™ Waste Disposal at ISU Iowa State University, Ames IA • Wastewater officials in Ames, Iowa approved the disposal of SYBR Safe™ as nontoxic waste • Diluted dye solution can be poured directly down the drain at Iowa State University ISU scientists can dispose SYBR Safe™ liquid waste down the drain 13

How is SYBR Safe™ better for my sample? 14

Sensitivity comparison Kb 23. 1 9. 4 6. 6 4. 4 2. 3 2. 0 Ethidium bromide SYBR Safe™ Marker is l DNA/Hind III Fragment SYBR Safe™ has comparable sensitivity to ethidium bromide 15

E-Gel® - Et. Br versus SYBR Safe™ 1. 2% E-Gel® with ethidium bromide 0. 3 second exposure Same samples and volumes loaded on both gels. Both gels viewed on UV transilluminator 1. 2% E-Gel® with SYBR Safe™ 1 second exposure Same sensitivity as ethidium bromide, but longer exposure Invitrogen Proprietary and Confidential

How does one use SYBR Safe™? 17

Using SYBR Safe™ Formats offered: • Offered as 10, 000 X concentrate, or • Ready-to-use solution in 1 X TAE • Ready-to-use solution in 0. 5 X TBE • E-Gel® pre-cast agarose gels incorporated with SYBR Safe™ How to use: • Incorporate into the gel, or • Perform post-run staining, or • Use E-Gel® with SYBR Safe™ is convenient to use 18

What does UV exposure do to DNA? 19

Effect of UV on DNA SYBR Safe. TM & Blue Light Time (Min) 2 4 8 16 Ethidium bromide & UV Light 2 4 8 16 Nicked DNA Supercoil DNA CFUs obtained from transformation with constructs containing inserts stained with SYBR Safe™ or ethidium bromide and viewed on blue light or UV respectively UV damages DNA and diminishes cloning results Invitrogen Proprietary and Confidential

Eliminate the dangers of UV transillumination Safe Imager Blue light Transilluminator • Does not damage your skin and eyes • Produces bright light and uniform emission • Provides optimal excitation for SYBR Safe™ DNA gel stain • For more information, visit www. invitrogen. com/safeimager Optimum excitation of SYBR Safe™ and maximum safety Invitrogen Proprietary and Confidential

Safe Imager versus the competition Competitor blue light box Safe Imager™ technology Even illumination with the Safe Imager™ Invitrogen Proprietary and Confidential

Safe Imager for visualizing stained proteins 25. 1 25 0. 1 2 4 8 16 32 64 12 8 25 6 50 0 SYPRO Ruby staining ng protein per band (approx. ) 2 x dilution series Mark 12 Markers Safe. Imager 1 ng sensitivity with SYPRO Ruby and Safe. Imager Invitrogen Proprietary and Confidential

Mark 12 standards 0. 125 0. 5 1 2 4 8 16 32 64 128 256 500 Safe Imager and SYPRO-Ruby Staining of 2 -D gel Jurkat cell lysate Avoid UV exposure when performing 2 -D electrophoresis Invitrogen Proprietary and Confidential

Lucy 565 (UV) 0. 125 0. 5 1 2 4 8 16 32 64 128 256 Safe Imager and SYPRO Orange protein stain SYPRO Orange (Safe Imager) Superior sensitivity with SYPRO Orange and Safe Imager versus Sigma’s Lucy 565 Invitrogen Proprietary and Confidential

Lucy 506 (Safe Imager) 0. 125 0. 5 1 2 4 8 16 32 64 128 256 0. 25 0. 125 0. 5 1 2 4 8 16 32 64 128 256 Safe Imager and SYPRO Ruby protein stain SYPRO Ruby (Safe Imager) Mark 12 standards Superior sensitivity with SYPRO Ruby and Safe Imager versus Sigma’s Lucy 506 Invitrogen Proprietary and Confidential

The complete safety solution SYBR Safe™ DNA Gel Stain • Better for you • Better for the environment • Better for your institution • Better for your sample All this with … • No compromise on performance • For more information, visit www. invitrogen. com/sybrsafe Safe Imager Blue light Transilluminator • Avoid UV exposure • Get improved cloning efficiency • Can be used with DNA and protein stains SYBR Safe™ and Safe Imager™ offer the compete safety solution Invitrogen Proprietary and Confidential

Information Learn more about: SYBR Safe™ at www. invitrogen. com/sybrsafe Safe Imager™ at www. invitrogen. com/safeimager Additional information or questions: E-mail sybrsafe@invitrogen. com We like you just the way you are! 28