SYBR Safe™ versus ethidium bromide

Nikita Warner, Ph.D.Assoc. Product ManagerNucleic Acid Separation & Analysis

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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

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How is SYBR Safe™ better for me?

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Oral toxicity evaluation

Displays no acute 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.** Peformed by Northview Pacific Laboratories, Inc., Hercules, CA.

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Genotoxicity studies

Displays greatly reduced genotocixity

In Vitro Test* Cell TypeResult 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).

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Ames test results

Causes fewer mutations in the standard Ames test

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How is SYBR Safe™ better for the environment?

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Hazardous waste testing

Not classified as hazardous waste under U.S. federal regulations

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.

According to the guidelines of the EPA, SYBR Safe™ DNA gel stain is not • corrosive• ignitable, or • reactive

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NPDES analysis

SYBR Safe™ DNA gel stain does not differ significantly from 0.5X TBE buffer

Test SYBR Safe Stain in 0.5X TBE 0.5X TBE

PH (150.1) 8.45 8.48

Total cynaide (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.

Tests performed according to National Pollutant Discharge Elimination System (NPDES) guidelines indicate that SYBR Safe™ DNA gel stain does not differ significantly from 0.5X TBE buffer

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Organic compound analysis

SYBR Safe™ DNA gel stain in 0.5X TBE is indistinguishable from 0.5X TBE alone

Tested were performed on 0.5X TBE and 1X SYBR Safe™ in 0.5X TBE for:

• Organochlorine pesticides and PCBs (608M)

• Volatile organic compounds (624)

• Semi-volatile organic compounds (625)

None of the organic pollutant compounds tested for were detected in either sample

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How is SYBR Safe™ better for my institution?

Invitrogen Proprietary and Confidential

SYBR Safe™ Institutional Savings

14% institutional savings at a Boston institute

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•$0.00Disposal costs - Time & Labor •$0.00Overhead disposal costs •None•Total SYBR Safe™ cost•$0.50 per gel

For more details, visit:www.invitrogen.com/sybrsafe

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SYBR Safe™ Waste Disposal at ISU

ISU scientists can dispose SYBR Safe™ liquid waste down the drain

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

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How is SYBR Safe™ better for my sample?

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Sensitivity comparison

SYBR Safe™ has comparable sensitivity to ethidium bromide

Ethidium bromide SYBR Safe™

Marker is DNA/Hind III Fragment

23.1

9.4

6.6

4.4

2.02.3

Kb

Invitrogen Proprietary and Confidential

E-Gel® - EtBr versus SYBR Safe™

1.2% E-Gel® with SYBR

Safe™1 second exposure

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

Same sensitivity as ethidium bromide, but longer exposure

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How does one use SYBR Safe™?

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Using SYBR Safe™

Formats offered:

• Offered as 10,000X concentrate, or

• Ready-to-use solution in 1X TAE

• Ready-to-use solution in 0.5X 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™

SYBR Safe™ is convenient to use

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What does UV exposure do to DNA?

Invitrogen Proprietary and Confidential

Effect of UV on DNA

UV damages DNA and diminishes cloning results

CFUs obtained from transformation with constructs containing inserts stained with SYBR Safe™ or ethidium bromide and viewed on blue light or UV respectively

Time (Min)

Supercoil DNA

Nicked DNA

Ethidium bromide & UV Light

SYBR SafeTM & Blue Light

2 4 8 16 2 4 8 16

Invitrogen Proprietary and Confidential

Eliminate the dangers of UV transillumination

Optimum excitation of SYBR Safe™ and maximum safety

• 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

Safe Imager Blue light Transilluminator

Invitrogen Proprietary and Confidential

Safe Imager versus the competition

Even illumination with the Safe Imager™

Competitor blue light box Safe Imager™ technology

Invitrogen Proprietary and Confidential

SafeImager

2x dilution series Mark 12 Markers

ng protein per band (approx.)

1 ng sensitivity with SYPRO Ruby and SafeImager

32 16 8 4 2 1 0.5

0.25

.125

256

128

64500

Safe Imager for visualizing stained proteins

SYPRO Ruby staining

Invitrogen Proprietary and Confidential

Safe Imager and SYPRO-Ruby Staining of 2-D gel

32

16

8 4 2 1 0.5

0.2

5

0.1

25

256

128

64500

Mark12 standards Jurkat cell lysate

Avoid UV exposure when performing 2-D electrophoresis

Invitrogen Proprietary and Confidential

Safe Imager and SYPRO Orange protein stain

Lucy 565 (UV) SYPRO Orange (Safe Imager)

32

16

8 4 2 1 0.5

0.2

5

0.1

25

25

6

12

8

64

32

16

8 4 2 1 0.5

0.2

5

0.1

25

25

6

12

8

64

Superior sensitivity with SYPRO Orange and Safe Imager versus Sigma’s Lucy 565

Invitrogen Proprietary and Confidential

SYPRO Ruby (Safe Imager)Lucy 506 (Safe Imager)

32

16

8 4 2 1 0.5

0.2

5

0.1

25256

128

64

32

16

8 4 2 1 0.5

0.2

50

.12

5256

128

64

Mark12 standards

Safe Imager and SYPRO Ruby protein stain

Superior sensitivity with SYPRO Ruby and Safe Imager versus Sigma’s Lucy 506

Invitrogen Proprietary and Confidential

The complete safety solution

SYBR Safe™ and Safe Imager™ offer the compete safety solution

Safe Imager Blue light Transilluminator

• Avoid UV exposure• Get improved cloning efficiency• Can be used with DNA and protein stains

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

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Information

We like you just the way you are!

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