Fluorescent Dyes and Assays for Genomics and Proteomics Research

GelRed™ & GelGreen™ Safe Nucleic Acid Gel Stains ... pp. 2-3 EvaGreen® Dye for qPCR ... pp. 3-6 PMA™ for bacterial viability ... p. 7 PMA-Lite™ photolysis device ... p. 7 AccuLite™ Handheld Fluorometers ... p. 8 AccuBlue™ and AccuClear™ DNA Quantitation ... p. 8-9 Lumitein™ Protein Gel Stain ... p. 10 AccuOrange™ Protein Quantitation Assay ... p. 11 GelRed™ & GelGreen™ Nucleic acid gel stainssensitive

elRed™ and GelGreen™ are next-generation fluorescent nucleic acid Superior sensitivity gel stains designed to replace the highly toxic (EtBr). G Developed by scientists at Biotium, GelRed™ and GelGreen™ are superior GelRed™ and GelGreen™ are highly sensitive for precast or post- to EtBr and other EtBr alternatives by having a combination of low toxicity, high electrophoresis gel . Designed primarily for use with a 312/302 nm UV sensitivity and exceptional stability. transilluminator, GelRed™ is much more sensitive than EtBr. GelGreen™ is compatible with visible blue light transilluminators and UV to blue light converter EtBr has been the predominant dye used for nucleic acid gel staining for plates, which protect users and DNA samples from UV light exposure. Using blue decades because of its low price and generally sufficient sensitivity. However, light excitation instead of UV when excising DNA from preparative gels greatly EtBr is a highly mutagenic chemical. The safety hazard and costs associated with improves downstream cloning efficiency (Gründemann and Schömig 1996). decontamination and waste disposal can ultimately make the dye expensive and GelGreen™ is spectrally similar to SYBR Safe®, but is far more sensitive than the inconvenient to use. For this reason, alternative gel stains, such as SYBR® dyes, latter. have become commercially available in recent years. While these alternative dyes have reduced mutagenicity, they sacrifice sensitivity and stability. For example, Another advantage of GelRed™ and GelGreen™ is their chemical stability. SYBR® Safe has very limited sensitivity while SYBR® Green and SYBR® Gold You can handle the two dyes the same way as EtBr. This means that the dyes are much less stable than EtBr. SYBR® dyes also enter cells rapidly to stain are perfectly stable in water at room temperature for long-term storage, and they mitochondria and nuclear DNA, making it more likely for the dyes to be harmful to can be added to molten for making precast gels. Both dyes are also very cells. Indeed, SYBR® Green I has been shown to strongly potentiate DNA mutation photostable and can be handled under normal room light. caused by UV light and other (Ohta, et al. 2001). Biotum gel extraction kits and DNA ladders are optimized for use with GelRed Safer options for gel staining and GelGreen precast gels. GelRed™ and GelGreen™ also are compatible with popular gel extraction kits and DNA ladders from other suppliers. To make safer gel stains, scientists at Biotium used a novel yet very simple concept: reducing genotoxicity by preventing the dyes from entering living cells. References We believe that the mutagenicity of a DNA-binding dye can greatly reduced by denying it access to genomic DNA in living cells. Thus, we engineered the Gründemann and Schömig. Protection of DNA During Preparative Agarose Against Damage Induced by Ultraviolet Light BioTechniques 21:898 (1996). chemical structures of GelRed™ and GelGreen™ such that the dyes are incapable of crossing cell membranes. Ames tests have confirmed that GelRed™ and Ohta, et al. Ethidium bromide and SYBR Green I enhances the genotoxicity of UV-irradiation and chemical mutagens in E. coli. Mut. Res 492, 91 (2001). GelGreen™ are nonmutagenic at concentrations well above the concentrations used for gel staining. Furthermore, environmental safety tests showed that GelRed™ and GelGreen™ are non-toxic to aquatic life. Because of this, GelRed™ and GelGreen™ are classified as non-hazardous waste, and can be disposed as regular trash or down the drain. For more information, please download the GelRed, GelGreen, and their uses are covered by US patent numbers 7,960,498 and 7,803943. GelRed™/GelGreen™ Safety Report at www.biotium.com. SYBR is registered trademark of Molecular Probes Corp.

EtBr GelRed™ SYBR® Safe GelGreen™ 1 kb and 100 bp DNA ladders

1 kb 100 bp bp • Ready-to-use at optimal 10,000 & 8,000 6,000 & 5,000 concentration for loading 4,000 3,000 GelRed™ and GelGreen™ 2,500 precast gels 2,000 bp 1,500 1,500 • 1 kb ladder: 250 bp-10 kb

1,000 1,000 • 100 bp ladder: 100 bp-1.5 kb 900 750 800 700 • Also available with loading 600 buffer suplied separately. 500 500 400 300 250 200 Figure 2. 100 ng each of 1 kb ladder 100 (left) and 100 bp ladder (right) run on 1% agarose/TBE gels containing 1X GelRed™. Gel was imaged with an EtBr Figure 1. GelRed™ and GelGreen™ are more sensitive than EtBr and SYBR® Safe. filter using a GelDocIt™ system from UVP. Left: Comparison of GelRed™ and ethidium bromide (EtBr) in precast gel staining using 1% agaose gel in TBE buffer. Right: Comparison of GelGreen™ and SYBR® Safe in post gel staining using 1% agarose gel in TBE buffer. Two-fold serial dilutions of 1 kb Plus DNA Ladder from Invitrogen were loaded onto each gel in 4 lanes in the amounts of 200 ng, 100 ng, 50 ng and 25 ng, respectively, from left to right.

2 Genomics and Proteomics Products • www.biotium.com • 800-304-5357 Safer, ultra-sensitive alternatives to EtBr and SYBR® dyes

1000 SYBR Green EtBr 750 GelGreen GelRed FEATURES 500 Safer than EtBr

Shown by Ames test and other tests to be of Colonies Number 250 nonmutagenic and noncytotoxic.

Easy disposal 0 0 0.1 0.5 1 5 10 25 50 Passed environmental safety tests for direct Dose (ug/plate) disposal down the drain or in regular trash. Figure 4. GelRed™ and GelGreen™ are non-mutagenic. Comparison of mutagenicity among GelGreen™, GelRed™, SYBR® Green I and EtBr in +1 frameshift Salmonella Ultra-sensitive indicator strain TA98 in the presence of S9 fraction. SYBR® Green I became cytotoxic at More sensitive than EtBr and SYBR® Safe. 50 ug/plate. For more information, download the GelRed™ and GelGreen™ Safety Report at www.biotium.com. Extremely stable Stable in solution at room temperature. Table 1. Ordering Information Description Cat. No. Unit Size Features Simple to use For precast or post-electrophoresis gel staining. 41003-T 25 uL Newer, safer GelRed™ formulation in water 41003 0.5 mL Compatible with standard instruments 10,000X in water (recommended for GelRed™ replaces EtBr; GelGreen™ replaces 41003-1 10 mL new users) SYBR® dyes. GelRed™ Original formulation in 41002 0.5 mL 10,000X in DMSO DMSO

GelRed™ Ready to use for 41001 4 L 3X in water post-staining

41005-T 25 uL Newer, safer GelGreen™ formulation in water 41005 0.5 mL 10,000X in water (recommended for 41005-1 10 mL new users) SYBR® Safe GelRed™ GelGreen™ GelGreen™ Original formulation in 41004 0.5 mL 10,000X in DMSO DMSO

Ready-To-Use 31022 150 lanes Optimal concentration 1 kb DNA Ladder for GelRed™/ Ready-To-Use GelGreen™ precast 31032 150 lanes 100 bp DNA Ladder gels*

1 kb DNA Ladder 31021 30 ug Stand-alone ladders 100 bp DNA Ladder 31031 30 ug

Figure 3. GelRed™ and GelGreen™ gel stains are safer because they cannot 31030-50 50 columns Validated for use penetrate cell membranes to bind DNA in living cells. HeLa cells were incubated DNA Gel Extraction Kit with GelRed™ and at 37 oC with 1X SYBR® Safe, GelGreen™ or GelRed™, respectively. Images 31030-250 250 columns GelGreen™* were taken following incubation with dye for 30 min. The top row shows phase contrast images of the field of cells, the bottom row shows green for Electrophoresis buffer SYBR® Safe and GelGreen™ and red fluorescence for GelRed™. SYBR® Safe 5X TBE Buffer 41006 4 L rapidly entered cells and stained nuclei. GelRed™ and GelGreen™ were unable concentrate to cross cell membranes, demonstrated by the absence of fluorescence staining. * GelRed™ and GelGreen™ also are compatible with popular gel extraction kits and DNA ladders from other suppliers.

Genomics and Proteomics Products • www.biotium.com • 800-304-5357 3 EvaGreen® Dye for qPCR

® vaGreen dye is a next-generation DNA-binding dye with features ideal for PCR master mixes featuring EvaGreen® dye and Cheetah™ Taq use in quantitative real-time PCR (qPCR) and other applications. Biotium scientists designed the dye with several essential dye properties in mind, Biotium offers stand-alone EvaGreen® dye and a variety of qPCR master E mixes. Fast EvaGreen® Master Mix is recommended for most qPCR targets, and including PCR inhibition, dye safety, stability, and fluorescence spectra of the dye. The results of our efforts is a dye superior to other dyes such as SYBR® Green I is supplied with a separate vial of ROX reference dye, so customers can add the for PCR and melt curve analysis. amount of ROX recommended for their qPCR instrument. Fast Plus EvaGreen® Master Mix is formulated for more challenging qPCR applications, and is Superior PCR performance formulated to be more tolerant of inhibitory factors and to reduce mis-priming and primer dimer formation. Fast Plus EvaGreen® Master Mixes are pre-formulated EvaGreen® dye binds to dsDNA via a novel “release-on-demand” with either no ROX, low ROX, or high ROX reference dye for use with different mechanism, which permits the use of a relatively high dye concentration without qPCR instruments. Fast Probe Master Mix does not contain DNA dye, and can be inhibiting PCR. The dye is constructed of two monomeric DNA-binding dyes linked used with TaqMan® probes and Fluidigm’s microfluidics-based qPCR platforms. by a flexible spacer. The dimeric dye randomly shifts between an inactive looped Fast Probe Master Mix is available with or without high ROX reference dye. conformation that does not bind DNA and an active conformation that becomes EvaGreen® dye and mixes are available in a range of sizes, including low cost trial fluorescent upon DNA binding. When the concentration of DNA is low, such as sizes. at the beginning of a PCR reaction, the inactive conformation is favored. As TM DNA concentration increases during amplification, the DNA-bound conformation Biotium’s qPCR master mixes feature Cheetah Taq, our proprietary becomes stabilized. This chemical equilibrium provides a unique mechanism to chemically-modified hot-start DNA polymerase. Unlike AmpliTaq Gold®, which TM continuously supply the active form of the dye from the “reserve” dye in looped takes 10 minutes or longer to activate, Cheetah Taq is fully recovered in 2 conformation as more DNA is synthesized by PCR. Consequently, EvaGreen® minutes with high activity, making it particularly suitable for fast PCR. Cheetah™ can be used at relatively high dye concentrations, resulting in improved sensitivity Taq is completely inactive at room temperature and free of DNA contamination. and more accurate melt curve analysis compared to other qPCR dyes such as This makes Cheetah™ Taq superior to antibody-based hotstart Taq, which SYBR® Green. EvaGreen® dye also is compatible with microfluidics-based qPCR is typically not completely inactive at room temperature and is prone to DNA platforms and isothermal DNA amplification methods. EvaGreen® is detected contamination due to the nature of antibody production. using the same instrument settings as SYBR® Green. Selected References An added benefit of EvaGreen® dye is that you can analyze your PCR Cheng, et al. Detection of hemi/homozygotes through heteroduplex formation in high-resolution melting products by gel electrophoresis without the need to use a DNA gel stain. The analysis. Anal Biochem 410, 158 (2011). EvaGreen® dye in the PCR reaction doubles as a DNA prestain, permitting direct Cheng, et al. Development of a single-tube multiplex real-time PCR for detection and identification of five visualization of DNA bands following electrophoresis. pathogenic targets by using melting-curve analysis with EvaGreen. Arch Virol DOI 10.1007/s00705-012- 1493-6 (2012). Goldmeyer, et al. Development of a novel one-tube isothermal reverse transcription thermophilic helicase- dependent amplification platform for rapid RNA detection. J Mol Diagn 9, 639 (2007). Khan, et al. Detection of aacA-aphD, qacEδ1, marA, floR, and tetA genes from multidrug-resistant bacteria: comparative analysis of real-time multiplex PCR assays using EvaGreen® and SYBR® Green I dyes, Molecular and Cellular Probes 25, 78 (2011). Li, et al. Primase-based whole genome amplification. Nucleic Acids Res 36, e79 (2008). Mao, et al. Characterization of EvaGreen Dye and the implication of its physicochemical properties for qPCR applications. BMC Biotechnology 7, 76 (2007). Figure 1. EvaGreen® dye binds to dsDNA via a “release-on-demand” mechanism. Ohta, et al. Ethidium bromide and SYBR Green I enhances the genotoxicity of UV-irradiation and chemical mutagens in E. coli, Mut Res 492, 91 (2001). Safer handling and disposal Schaerli, et al. Isothermal DNA amplification using the T4 replisome: circular nicking endonuclease-dependent amplification and primase-based whole-genome amplification. NucleicAcids Res 38, e201 (2010). A unique feature of EvaGreen® dye is its safety. The toxicity and mutagenicity of PCR dyes has not been thoroughly studied, despite the increasing White, et al. Methylation-sensitive high-resolution melt-curve analysis of the SNRPN gene as a diagnostic screen for Prader-Willi and Angelman Syndromes. Clin Chem 53, 1960 (2007). use of qPCR in research and diagnostics. Thus, handling and disposal of qPCR master mixes may pose health and environmental risks. For example, SYBR® Green I, a popular PCR dye, has been found to be even more environmentally toxic than ethidium bromide, a well-known (Ohta et al., 2001). SYBR® Green I has been suggested to interfere with the natural DNA-repair mechanism in cells and consequently potentiate DNA damage by mutagenic chemicals and UV light. With this in mind, Biotium’s scientists designed EvaGreen® dye such that it cannot cross cell membranes, thus preventing the dye from binding genomic DNA in living cells. Independent labs have confirmed that EvaGreen® dye is EvaGreen is a registered trademark of Biotium, Inc. EvaGreen dye technologies are covered by US nonmutagenic, noncytotoxic and safe to aquatic life. EvaGreen® dye is classified patent Nos. 7,601,498, 7,776,567 and other pending US and international patents. Amplitaq Gold and TaqMan are registered trademarks of Roche Molecular Systems, Inc. SYBR is a registered trademark as non-hazardous waste under California CCR Title 22, allowing disposal in of Invitrogen Corp. Practicing HRM may require a license from Idaho Technologies, Inc. regular trash or down the drain. A detailed safety report for EvaGreen® is available for download from www.biotium.com.

4 Genomics and Proteomics Products • www.biotium.com • 800-304-5357 Environmentally Friendly Dye with Superior qPCR Performance

FEATURES

Safer and more environmentally friendly EvaGreen® dye, which has passed California environmental regulation (CCR Title 22) for disposal down the drain.

High sensitivity Unique "release-on-demand" DNA binding mechanism of EvaGreen® dye enables superior PCR and melt curve analysis.

Figure 2. EvaGreen® dye for qPCR offers superior sensitivity compared to SYBR® Green. Comparison of Fast EvaGreen® master mix from Biotium and two fast Rapid hot-start SYBR® Green master mixes from two leading companies (company A and company Novel chemically-modified hot start Taq, Q) under similar conditions. The inset shows an enlarged view of the area near the baseline. Amplicon: ATPG fragment amplified from human genomic DNA; instrument: Cheetah™ Taq requires only 2 minutes to ABI 7900 Fast. activate.

Compatible with both fast and regular cycling protocols SYBR® Green I EvaGreen® Incubation Time Direct visualization of PCR product in gels Analyze your PCR product by gel electrophoresis 5 min. without the need to add another gel stain.

30 min.

Figure 3. EvaGreen® dye is safer because it cannot penetrate cell membranes to bind DNA in living cells. Comparison of cell membrane permeability between EvaGreen® dye and SYBR® Green I. HeLa cells were incubated with SYBR® Green I or EvaGreen® dye (1.2 uM final concentration for both dyes) at 37 oC. SYBR® Green I rapidly entered cells and stained nuclei, while GelGreen™ was unable to penetrate cell membranes, demon- strated by the absence of nuclear staining. For more information, download the EvaGreen Safety Report at www.biotium.com.

100 Cheetah Taq 80 AmpliTaq Gold

60

40 Cell-membrane impermeable ® Relative Activity (%) Activity Relative EvaGreen dye is the only PCR 20 dye to date that is non-mutagenic, 0 0 1 2 5 10 20 non-cytotoxic, and non-toxic to Minutes at 95°C aquatic life Figure 4. Cheetah™ Taq requires only 2 minutes of hot-start for full recovery of activity. Comparison of hot-start recovery of polymerase activity for Cheetah™ Taq and AmpliTaq Gold® following incubation at 95°C in 50 mM pH 8.0 Tris.

Genomics and Proteomics Products • www.biotium.com • 800-304-5357 5 EvaGreen® Dye, Master Mixes, and Related Products for qPCR

EvaGreen Dye Products for qPCR Product Name Features Fast Plus EvaGreen® qPCR Master Mix Suitable for both qPCR and melt curve analysis, the master mix contains EvaGreen® dye and our superior chemically modified hot start Cheetah™ Taq to deliver unmatched results. Does not contain Rox reference dye. Fast Plus EvaGreen® qPCR Master Mix, Fast Plus Master Mix with low concentration of Rox reference dye for use with ABI 7500, 7500 Fast or Stratagene Low Rox MX4000P, MX3000P, MX3005P instruments. Fast Plus EvaGreen® qPCR Master Mix, Fast Plus Master Mix with high concentration of Rox reference dye for use with ABI 5700, 7000, 7300, 7700, 7900, High Rox 7900HT, 7900HT Fast, StepOne, or StepOne plus instruments. Fast Probe Master Mix Contains our fast-activating hot start Cheetah™ Taq and proprietary buffer without DNA binding dye. Suitable for use with TaqMan®-like probes and Fluidigm® qPCR platforms. Fast Probe Master Mix, High Rox Contains our fast-activating hot start Cheetah™ Taq and proprietary buffer without DNA binding dye. Suitable for use with TaqMan®-like probes and Fluidigm® qPCR platforms. With high concentration of Rox reference dye. Evagreen™ Dye, 20X in water Stand-alone EvaGreen dye for use with your own PCR reaction mix. Cheetah™ Taq Stand-alone chemically-modified hot start Taq polymerase for use with your own PCR reaction mix. ROX Reference Dye, 25 uM in TE Stand-alone ROX reference dye for use with your own PCR reaction mix.

Ordering Information Description Size Cat. No. Unit Size Trial size, 100 reactions 31003-T Each (1 mL) 200 reactions 31003 Each (2 x 1 mL) Fast EvaGreen® qPCR Master Mix 500 reactions 31003-1 Each (5 x 1 mL) 5000 reactions 31003-2 Each (50 x 1 mL) Trial size, 100 reactions 31020-T Each (1 mL) 200 reactions 31020 Each (2 x 1 mL) Fast Plus EvaGreen® qPCR Master Mix 500 reactions 31020-1 Each (5 x 1 mL) 5000 reactions 31020-2 Each (50 x 1 mL) Trial size, 100 reactions 31014-T Each (1 mL) 200 reactions 31014 Each (2 x 1 mL) Fast Plus EvaGreen® qPCR Master Mix, Low ROX 500 reactions 31014-1 Each (5 x 1 mL) 5000 reactions 31014-2 Each (50 x 1 mL) Trial size, 100 reactions 31015-T Each (1 mL) 200 reactions 31015 Each (2 x 1 mL) Fast Plus EvaGreen® qPCR Master Mix, High ROX 500 reactions 31015-1 Each (5 x 1 mL) 5000 reactions 31015-2 Each (50 x 1 mL) Trial size, 100 reactions 31005-T Each (1 mL) 200 reactions 31005 Each (2 x 1 mL) Fast Probe Master Mix 500 reactions 31005-1 Each (5 x 1 mL) 5000 reactions 31005-2 Each (50 x 1 mL) Fast Probe Master Mix, High ROX Trial size, 100 reactions 31016-T Each (1 mL) 200 reactions 31016 Each (2 x 1 mL) 500 reactions 31016-1 Each (5 x 1 mL) 5000 reactions 31016-2 Each (50 x 1 mL) EvaGreen® Dye, 20X in Water, Trial Size 1 mL 31000-T Each (1 mL) EvaGreen® Dye, 20X in Water 5 x 1 mL 31000 Each (5 x 1 mL) Cheetah™ Taq 500 U 29050 Each (500 U) ROX Passive Reference Dye, 25 uM in TE 5 x 1 mL 29052 Each (5 x 1 mL)

6 Genomics and Proteomics Products • www.biotium.com • 800-304-5357 Selective Detection of Viable Bacteria with PMA™ and qPCR

B he ability to selectively and sensitively detect viable bacteria in the presence A of dead bacteria is of vital importance for many practical applications, 1.2 16 including safety inspection of food products, drinking water quality control, 14 T 1.0 12 and medical diagnosis. The traditional detection method based on bacterial culturing is time-consuming and insensitive. Detection based on PCR is a rapid 0.8 10 8

Killed, - PMA w/o PMA) and highly sensitive alternative method. However, PCR methods cannot distinguish 0.6 Live, + PMA - 6 live from dead cells, making interpretation of any analytical results difficult.The 0.4 Live, - PMA 4 novel DNA-modifying dye propidium monoazide (PMA™) developed by Biotium Killed, + PMA (with Ct 2 Normalized fluorescence 0.2 0 overcomes this problem. Pre-treating a sample with PMA™ prior to PCR analysis NTC - Ct w/o PMA Ct with PMA 0.0 -2 permits one to selectively detect only viable bacteria in a highly sensitive and 5 10 15 20 25 30 35 40 Controllive E. E.coli coli Killedkilled E. E. coli coli reliable manner. Cycle number Figure 2. Effect of PMA on quantitative PCR of control and heat-killed E. coli. (A) Representative PMA™ is a photo-reactive dye with a high affinity for DNA. The dye qPCR amplification curves for live and dead E. coli, untreated or treated with PMA. PCR was intercalates into dsDNA and forms a covalent linkage upon exposure to intense performed on purified genomic DNA with primers against a region of 16S rRNA. NTC: no template control. (B) The DCt of control and heat-killed E. coli with and without PMA treatment. The Ct value visible light, resulting in chemically modified DNA, which cannot be amplified by of sample without PMA was subtracted from the corresponding sample with PMA cross-linking (Ct PCR (Figure 1). Because PMA™ is designed to be cell membrane-impermeable, with PMA – Ct without PMA). when a sample comprising both live and dead bacteria is treated with PMA™, only dead bacteria are susceptible to DNA modification due to compromised cell membranes. Thus, subsequent DNA extraction followed by qPCR permits selective detection of live cells. The number of dead bacteria in a sample is proportional to the delay in PCR threshold cycle of a PMA-treated sample compared to the threshold cycle of an untreated sample. Conventional PCR and gel analysis can be performed with PMA, as well, using the amount of DNA amplified by the PCR PMA-Lite™ LED Photolysis Device reaction as an indicator of the number of viable cells in the sample. • Specifically designed for photoactivation of PMA, EMA and other dyes PMA™-qPCR method also have been used with non-bactieral organisms • Provides even illumination to up to 18 1.5-2 mL-sized vials. such as yeast, fungi, amoebae, and viruses. • Internal fan ensures a temperature of <37OC. Selected References: • Four timer settings for 10, 15, 20 or 30 minutes of illumination.

Andorra, et al. Determination of viable wine yeast using DNA binding dyes and quantitative PCR. Int J Food • Long-lasting LEDs with 465-475 nm emission Microbiol 144, 257 (2010). • Free vial of PMA with purchase Fittipaldi, et al. Discrimination of viable Acanthamoeba castellani trophozoites and cysts by propidium monoazide real-time polymerase chain reaction. J Eukaryot Microbiol 58, 359 (2011). Kobayashi, et al. Improving clinical significance of PCR: use of propidium monoazide to distinguish viable from dead Staphylococcus aureus and Staphylococcus epidermidis. J Orthop Res 27, 1243 (2009). Luo, et al. Method to detect only viable cells in microbial ecology. Appl Microbiol Biotechnol 86, 377 (2009). Nocker, et al. Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J Microbiol Methods 67, 310 (2006). Parshionikar, et al. Use of Propidium Monoazide in Reverse Transcriptase PCR To Distinguish between Infectious and Noninfectious Enteric Viruses in Water Samples. Applied Environmental Microbiol 76, 4318 (2010). Varma, et al. Quantitative real-time PCR analysis of total and propidum monoazide-resistant fecal indicator bacteria in wastewater. Water Res 43, 4790 (2009). Vesper, et al. Quantifying fungal viability in air and water samples using quantitative PCR after treatment with propidium monoazide (PMA). J Microbiol Methods 72, 180 (2008).

Figure 3. PMA-Lite™ LED Photolysis Device.

Ordering Information Description Cat. No. Unit Size PMA™ dye 40013 1 mg PMA™ dye, 20 mM in dH O 40019 100 uL Figure 1. Principle of PMA™-qPCR assays. 2 PMA-Lite™ LED Photolysis Device E90002 Each

Genomics and Proteomics Products • www.biotium.com • 800-304-5357 7 AccuBlue™ and ™ dsDNA Quantitation Assays

ccuBlue™ and AccuClear™ dsDNA quantitation assays allow precise quantitation of purified dsDNA samples across a wide range of concentrations. Unlike absorbance-based nucleic acid quantitation, fluorescent DNA binding dyes are highly sensitive and selective for double-stranded DNA and provide a more Aaccurate DNA concentration in the presence of contaminating RNA and other common contaminants including free nucleotides, protein, detergents and salts. Biotium offers three dsDNA quantitiation kits for different instruments and sample concentration ranges. The kits also differ in their tolerance of various contaminants; download the product protocols at www.biotium.com for details. The AccuClear™ Ultra High Sensitivity assay is based on a novel, next-generation DNA binding dye which offers unrivaled sensitivity and dynamic range compared to other DNA dyes. The kits are compatible with Biotium’s compact and affordable AccuLite™ Mini Fluorometers.

AccuBlue™ Broad Range • Linear range: 2-2000 ng dsDNA AccuLite™ Mini Fluorometers • Blue fluorescence (Ex/Em: 350/460 nm) • Accept 200 μL PCR tubes • Assay can be extended to 4000 ng dsDNA with minor loss of linearity • LCD touch-screen display • Blue fluorescence detection, compatible with • USB interface for data management fluorescence microplate reader and Biotium’s • Compact (185 x 90 x 35 mm, 10 oz) AccuLite™ 350 handheld fluorometer • Greater than 6 logs of dynamic range • Include programs for use with AccuBlue and AccuClear dsDNA quantitation assays AccuBlue™ High Sensitivity • Power Supply: 4 AA batteries or a 5V DC adapter • Linear range: 0.2-100 ng dsDNA • AccuLite 350: 365-370 nm LED excitation 460+/-20 nm emission • Green fluorescence (Ex/Em: 485/530 nm) • AccuLite 470: 465-475 nm LED excitation • Membrane-impermeable dye is non-toxic and 540+/-30 nm emission non-mutagenic, for safer handling and easy disposal • Green fluorescence detection compatible with fluorescence microplate reader, NanoDrop® fluorospectrophotometer, Biotium’s AccuLite™ 470 handheld fluorometer and other handheld fluorometers like Qubit® and QuantiFluor-P™

AccuClear™ Ultra High Sensitivity • Linear range: 0.03-250 ng dsDNA • Green fluorescence (Ex/Em: 460/507 nm) • Unrivaled sensitivity and dynamic range • Novel green fluorescent dye is a perfect match for blue LED excitation sources • Green fluorescence detection compatible with fluorescence microplate reader, Biotium’s AccuLite™ 470 handheld fluorometer, or Figure 1. AccuLite™ Mini Fluorometers NanoDrop® fluorospectrometer

8 Genomics and Proteomics Products • www.biotium.com • 800-304-5357 Superior sensitivity and broad dynamic range

dsDNA-Selective Assays AccuBlue™ Broad Range (2-2000 ng) 7000 1200 75 6000 1000 dsDNA 50 5000 25

800 Fluorescence 0 4000 0 5 10 15 20 DNA (ng/well) R² = 0.9979 600

Fluorescence 3000 Fluorescence 400 2000 RNA

200 1000 ssDNA 0 0 0 20 40 60 80 100 0 500 1000 1500 2000 DNA/RNA (ng/well) DNA (ng/well) Figure 2: Triplicate samples of calf thymus dsDNA, mouse liver RNA or Figure 3: Standard curve of calf thymus DNA assayed using AccuBlue Broad salmon sperm ssDNA were assayed with the AccuBlue High Sensitivity Range Kit and read on a microplate reader (Ex/Em 350/460). Inset shows dsDNA Quantitation Kit. The AccuBlue High Sensitivity Assay is selective the lower end of the titration. for double-stranded DNA over RNA or single-stranded DNA.

AccuBlue™ Broad Range vs. Quant-iT™ Broad Range (2-4000 ng) AccuBlue™ High Sensitivity (0.2-100 ng)

9000 2500 100 75 2000 50 25

6000 AccuBlue™ Fluorescence 0 1500 0 1 2 3 4 5 DNA (ng/well) R² = 0.9994

Fluorescence 1000 Fluorescence 3000 Quant-iT™ 500

0 0 0 20 40 60 80 100 0 1000 2000 3000 4000 DNA (ng/well) DNA (ng/well)

Figure 4: Two-fold dilutions of calf thymus DNA were assayed using AccuBlue Figure 5: Standard curve of calf thymus DNA assayed using the AccuBlue or Quant-iT Broad Range assay kits. AccuBlue has improved linearity and High Sensitivity Kit and read on a microplate reader (Ex/Em 485/530). Inset wider dynamic range than the Quant-iT Broad Range. shows the lower end of the titration.

AccuClear™ Ultra High Sensitivity 0.03-250 ng) AccuBlue™ 14000 ™ 60 HS Dye PicoGreen Dye 12000 40

10000 20

Fluoresence 0 8000 0 0.2 0.4 0.6 0.8 1 R² = 0.998 DNA ng/well 6000 Fluoresence

4000

2000

0 0 50 100 150 200 250 DNA ng/well Figure 7: The AccuBlue High Sensitivity (HS) reagent and PicoGreen were diluted to working concentrations in HeLa cell cultures and incubated for 30 minutes. Figure 6: Standard curve of calf thymus DNA assayed using the AccuClear PicoGreen and the Quant-iT High Sensitivity reagent (Invitrogen) readily bind Ultra High Sensitivity Kit and read on a microplate reader (Ex/Em 460/507). nuclear DNA while no nuclear staining is evident with the AccuBlue HS reagent, Inset shows the lower end of the titration. illustrating its overall safety and lower toxicity due to its membrane impermeability.

Ordering Information Linear Range Description Cat. No. Unit Size AccuBlue Broad Range dsDNA Quantitation Solution, Trial Size 31009-T Each, 200 Assays Broad Range AccuBlue Broad Range dsDNA Quantitation Solution 31009 Each, 1000 Assays 2-2000 ng AccuBlue Broad Range dsDNA Quantitation Kit with 9 DNA standards 31007 Each, 1000 Assays AccuBlue High Sensitivity dsDNA Quantitation Solution, Trial Size 31008-T Each, 200 Assays High Sensitivity AccuBlue High Sensitivity dsDNA Quantitation Solution 31008 Each, 1000 Assays 0.2-100 ng AccuBlue High Sensitivity dsDNA Quantitation Kit with 8 DNA Standards 31006 Each, 1000 Assays AccuClear Ultra High Sensitivity dsDNA Quantitation Solution, Trial Size 31027-T Each, 200 Assays Ultra High Sensitivity AccuClear Ultra High Sensitivity dsDNA Quantitation Solution 31027 Each, 1000 Assays AccuClear Ultra High Sensitivity dsDNA Quantitation Kit with 7 DNA Standards 31028 Each, 1000 Assays 0.03-250 ng AccuClear Ultra High Sensitivity dsDNA Quantitation Kit with 1 DNA Standard 31029 Each, 4000 Assays AccuLite Mini AccuLite 350 Mini Fluorometer E90000 Each Fluorometers AccuLite 470 Mini Fluorometer E90001 Each

Genomics and Proteomics Products • www.biotium.com • 800-304-5357 9 Lumitein™ Protein Gel Stain Ultra Sensitive, Rapid PAGE Gel Staining

A B

FEATURES ~ 0.2 ng Highly sensitive At least as sensitive as silver stain, allowing detection of as little as 1 ng protein or less (Figure 1A). Figure 1. A. Two-fold serial dilutions of Precision Plus Protein™ standards (Bio-Rad) were separated via SDS-PAGE and then stained with LumiteinTM for 90 minutes without a separate fixation step. Gels were imaged on a GE Typhoon Trio™ scanner using 532 nm excitation and Simple & fast staining 610 BP 30 emission filter. B. 2-D gel of human liver protein lysate stained with Lumitein™ and imaged on a GE Typhoon Trio™ scanner. The three circled spots were excised and subjected Fixation and staining is combined in a single to mass spectroscopy (MS) analysis by Applied Biomics, Inc. (Hayward, CA). The results step, for results in 30-90 minutes. Gels can be confirmed that Lumitein™ gel staining is fully compatible with MS analysis (data not shown). left in staining solution longer than 90 minutes without overstaining. Native gels can be stained after a brief soak in SDS/acetic acid. Table 1. Comparison of standard staining protocols Protocol SYPRO® Ruby Lumitein™ Compatible with common instruments Step Fixation 15 min. 50% methanol/ Image using a UV light box, Dark Reader®, or None laser scanner. step 1 7.5% acetic acid Fixation 15 min. 50% methanol/ None Wide linear detection range step 2 7.5% acetic acid

At least three orders of magnitude. Staining Overnight 30-90 min.

Compatible with downstream analysis No destaining required Compatible with mass spectroscopy and 30 min. 10% methanol/ Destaining Optional: 5 min. in 30% Edman peptide sequencing (Figure 1B). 7% acetic acid methanol/ 15% acetic acid or 20 min. in water Economical and convenient Rinse 1 5 min. water Available as a 100X concentrated solution to Single 5 min. water rinse reduce manufacturing cost and shipping cost, Rinse 2 5 min. water or as a ready-to-use 1X staining solution for convenience. Total time More than 16 hours 2 hours or less

Highly stable Ordering Information 100X concentrate and 1X working solution are stable at room temperature for at least 1 year. Description Cat. No. Size 21001 200 mL Lumitein™ Protein Gel Stain, 1X 21001-1 1 L 21001-2 5 x 1 L 21002 2 mL Selected References Lumitein™ Protein Gel Stain, 100X 21002-1 10 mL Mishima, et al. Hinge and Chromoshadow of HP1alpha Participate in Recognition of K9 Methylated Histone H3 in Nucleosomes. J Mol Biol DOI:10.1016/j.jmb.2012.10.018 (2012). 21002-2 50 mL Troll, et al. Taming the symbiont for coexistence: a host PGRP neutralizes a bacterial symbiont. Environ Microbiol 12, 2190 (2010). Vera Pingitore, et al. Influence of vitamins and osmolites on growth and bacteriocin production by Lactobacillus salivarius CRL 1328 in a chemically defined medium. Can J Microbiol 55, 304 (2009). Lumitein and its related technologies are covered by pending US and international patents. Lumitein Vera Pingitore, et al. Characterization of salivaricin CRL 1328, a two-peptide bacteriocin produced is a trademark of Biotium, Inc. Dark Reader is a registered trademark of Clare Chemical Research; by Lactobacillus salivarius CRL 1328 isolated from the human vagina. Research in Microbiology ImageQuant and Typhoon Trio are trademarks of GE Healthcare; Precision Plus Protein is a 160, 401 (2009). trademark of Bio-Rad Laboratories; SYPRO is a registered trademark of Molecular Probes, Inc.

10 Genomics and Proteomics Products • www.biotium.com • 800-304-5357 AccuOrange™ Protein Quantitation Kit Highly sensitive, fluorometric protein assay

AccuOrange™ Protein Quantitation Kit is a highly sensitive fluorescence-based assay for quantitating purified protein samples in 96-well format.The detection range of the assay is 0.1-15 ug/mL protein. AccuOrange is much more sensitive than traditional protein quantitation assays such as BCA, Bradford and Lowry, and shows superior linearity and reproducibility compared to the NanoOrange® protein quantitation assay (Figure 2). The assay shows minimal variability between different proteins, and has stable fluorescence signal for up to 16 hours.

AccuOrange is recommended for quantitating purified protein or antibody samples.The tolerance of the AccuOrange assay to salts, buffers, detergents, and other chemicals is shown in Table 1. The AccuOrange assay has low tolerance for non-ionic detergents, and is not recommended for use with cell lysates containing Triton X-100, sodium deoxycholate, CHAPs, or other non-ionic detergents. The assay can tolerate up to 0.01% SDS (final concentration in assay).

Table 1. Comparison of AccuOrange™ with other protein quantitation assays Detection Range Assay Type Comments FEATURES (microplate assay) • Fluorescence detection (480/598 nm) • Ex/Em: 480/598 nm • Highly linear AccuOrange™ 0.1-15 ug/mL • Signal stable for at least 16 hours • Linear detection range: 0.1-15 ug/mL protein • Compatible with reducing agents • 200 uL microplate assay • Not compatible with detergents • Fluorescence detection (470/570 nm) • Minimal protein-protein variation • Non-linear • Fluorescence signal stable for at least 16 hours NanoOrange® 0.1-10 ug/mL • Fluorescence stable for 6 hours • Compatible with reducing agents • Compatible with reducing agents, amino acids, • Not compatible with detergents nucleic acids, and imidazole • Absorbance detection (750 nm) • For use with purified protein or antibody samples • Non-llinear Modified Lowry 1-1500 ug/mL • Not compatible with reducing agents • Compatible with AccuLite™ 470 Mini-Fluorometer • Not compatible with detergents (see p. 8) • Absorbance detection (562 nm) • Highly linear BCA 20-2000 ug/mL • Signal not stable over time • Not compatible with reducing agents • Compatible with detergents • Absorbance detection (595 nm) • Signal not stable over time Bradford 50-500 ug/mL • Non-linear (Coomassie) 2500 • Compatible with reducing agents 400 • Not compatible with detergents

2000 200 • Absorbance detection (660 nm) NanoOrange • Non-linear R² = 0.9453 Pierce® 660 nm 50-2000 ug/mL Fluorescence 0 • Compatible with reducing agents 1500 0 0.5 1 1.5 BSA (ug/mL) • Compatible with detergents • Absorbance detection (280 nm) 1000 AccuOrange • High protein-protein variability Fluorescence R² = 0.9981 A 50-2000 ug/mL 280 • Contaminants such as nucleic acids can affect results 500 AccuOrange NanoOrange NanoOrange is a registered trademark of Molecular Probes Inc. 0 0 5 10 15 BSA (ug/mL)

Figure 1. AccuOrange™ shows better linearity and reproducibility compared to NanoOrange® Protein Quantitation assay. BSA titration was performed in triplicate using AccuOrange Protein Ordering Information Quantitation Kit or NanoOrange Protein Quantitation Kit from Life Technologies according to manufacturer’s protocol and read on a microplate reader at the recommended wavelengths for Description Cat. No. Size each assay. Inset shows the lower end of the curve. Error bars represent standard deviation of the mean for triplicate samples. 30071-T 200 assays AccuOrange™ Protein Quantitation Kit 30071 2000 assays

Genomics and Proteomics Products • www.biotium.com • 800-304-5357 11 Glowing Products for Science™ www.biotium.com

Biotium, Inc. 3159 Corporate Place, Hayward, CA 94545