Use of SYBR Green I for Rapid Epifluorescence Counts of Marine Viruses and Bacteria
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Gelred® and Gelgreen® Safety Report
Safety Report for GelRed® and GelGreen® A summary of mutagenicity and environmental safety test results from three independent laboratories for the nucleic acid gel stains GelRed® and GelGreen® www.biotium.com General Inquiries: [email protected] Technical Support: [email protected] Phone: 800-304-5357 Conclusion Overview GelRed® and GelGreen® are a new generation of nucleic acid gel stains. Ethidium bromide (EB) has been the stain of choice for nucleic acid gel They possess novel chemical features designed to minimize the chance for staining for decades. The dye is inexpensive, sufficiently sensitive and very the dyes to interact with nucleic acids in living cells. Test results confirm that stable. However, EB is also a known powerful mutagen. It poses a major the dyes do not penetrate latex gloves or cell membranes. health hazard to the user, and efforts in decontamination and waste disposal ultimately make the dye expensive to use. To overcome the toxicity problem In the AMES test, GelRed® and GelGreen® are noncytotoxic and of EB, scientists at Biotium developed GelRed® and GelGreen® nucleic acid nonmutagenic at concentrations well above the working concentrations gel stains as superior alternatives. Extensive tests demonstrate that both used in gel staining. The highest dye concentrations shown to be non-toxic dyes have significantly improved safety profiles over EB. and non-mutagenic in the Ames test for GelRed® and GelGreen® dyes are 18.5-times higher than the 1X working concentration used for gel casting, and 6-times higher than the 3X working concentration used for gel staining. This Dye Design Principle is in contrast to SYBR® Safe, which has been reported to show mutagenicity At the very beginning of GelRed® and GelGreen® development, we made a in several strains in the presence of S9 mix (1). -
Supplementary Data for Publication
Electronic Supplementary Material (ESI) for Physical Chemistry Chemical Physics. This journal is © the Owner Societies 2016 Supplementary Data for Publication Synthesis of Eucalyptus/Tea Tree Oil Absorbed Biphasic Calcium phosphate-PVDF Polymer Nanocomposite Films: A Surface Active Antimicrobial System for Biomedical Application Biswajoy Bagchi1,δ, Somtirtha Banerjee1, Arpan Kool1, Pradip Thakur1,2, Suman Bhandary3, Nur Amin Hoque1 , Sukhen Das1+* 1Physics Department, Jadavpur University, Kolkata-700032, India. 2Department of Physics, Netaji Nagar College for Women, Kolkata-700092, India. 3Division of Molecular Medicine, Bose Institute, Kolkata-700054, India. +Present Address: Department of Physics, Indian Institute of Engineering Science and Technology, Shibpur, Howrah, West Bengal-711103, India. §Present Address: Fuel Cell and Battery Division, Central Glass and Ceramic Research Institute, Kolkata-700032, India. *Corresponding author’s email id: [email protected] Contact: +919433091337 Antimicrobial activity of EU and TTO treated films on E .coli and S. aureus by acridine orange/ethidium bromide (AO/EB) dual staining Live/dead cell characterization of EU/TTO film treated bacterial cultures was also done to visualize the viability under fluorescence microscope (). The treated culture suspensions after 12 and 24 hours of incubation were collected by centrifugation (5000 rpm, 20 mins). The cell pellets were resuspended in PBS. The staining solution was prepared by mixing equal parts of acridine orange (5mg/mL) and ethidium bromide (3mg/mL) in ethanol. 20μL of the staining solution is then mixed with 10μL of the resuspended solution and incubated for 15 minutes at 37°C. 10μL of this solution was then placed on a glass slide and covered with cover slip to observe under fluorescence microscope. -
Coming-Of-Age Characterization of Soil Viruses: a User's Guide To
Review Coming-of-Age Characterization of Soil Viruses: A User’s Guide to Virus Isolation, Detection within Metagenomes, and Viromics Gareth Trubl 1,* , Paul Hyman 2 , Simon Roux 3 and Stephen T. Abedon 4,* 1 Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA 94550, USA 2 Department of Biology/Toxicology, Ashland University, Ashland, OH 44805, USA; [email protected] 3 DOE Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; [email protected] 4 Department of Microbiology, The Ohio State University, Mansfield, OH 44906, USA * Correspondence: [email protected] (G.T.); [email protected] (S.T.A.) Received: 25 February 2020; Accepted: 17 April 2020; Published: 21 April 2020 Abstract: The study of soil viruses, though not new, has languished relative to the study of marine viruses. This is particularly due to challenges associated with separating virions from harboring soils. Generally, three approaches to analyzing soil viruses have been employed: (1) Isolation, to characterize virus genotypes and phenotypes, the primary method used prior to the start of the 21st century. (2) Metagenomics, which has revealed a vast diversity of viruses while also allowing insights into viral community ecology, although with limitations due to DNA from cellular organisms obscuring viral DNA. (3) Viromics (targeted metagenomics of virus-like-particles), which has provided a more focused development of ‘virus-sequence-to-ecology’ pipelines, a result of separation of presumptive virions from cellular organisms prior to DNA extraction. This separation permits greater sequencing emphasis on virus DNA and thereby more targeted molecular and ecological characterization of viruses. -
Marine Microplankton Ecology Reading
Marine Microplankton Ecology Reading Microbes dominate our planet, especially the Earth’s oceans. The distinguishing feature of microorganisms is their small size, usually defined as less than 200 micrometers (µm); they are all invisible to the naked eye. As a group, sea microbes are extremely diverse, and extremely versatile with respect to their abilities to make and eat food. All marine microbes are too small to swim against the current and are therefore classified as plankton. First we will discuss several ways to classify marine microbes. 1. Size Planktonic marine organisms can be divided into the following size categories: Category Size femtoplankton <0.2 µm picoplankton 0.2-2 µm nanoplankton 2-20 µm microplankton 20-200 µm mesoplankton 200-2000 µm In this laboratory we are concerned with the microscopic portion of the plankton, less than 200 µm. These organisms are not visible to the naked eye (Figure 1). Figure 1. Size classes of marine plankton 2. Type A. Viruses Viruses are the smallest and simplest microplankton. They range from 0.01 to 0.3 um in diameter. Externally, viruses have a capsid, or protein coat. Viruses can also have simple or complex external morphologies with tail fibers and structures that are used to inject DNA or RNA into their host. Viruses have little internal morphology. They do not have a nucleus or organelles. They do not have chlorophyll. Inside a virus there is only nucleic acid, either DNA or RNA. Viruses do not grow and have no metabolism. Marine viruses are highly abundant. There are up to 10 billion in one liter of seawater! B. -
Marine Microbiology at Scripps
81832_Ocean 8/28/03 7:18 PM Page 67 Special Issue—Scripps Centennial Marine Microbiology at Scripps A. Aristides Yayanos Scripps Institution of Oceanography, University of California, • San Diego, California USA Marine microbiology is the study of the smallest isolated marine bioluminescent bacteria, isolated and organisms found in the oceans—bacteria and archaea, characterized sulfate-reducing bacteria, and showed many eukaryotes (among the protozoa, fungi, and denitrifying bacteria could both produce and consume plants), and viruses. Most microorganisms can be seen nitrous oxide, now known to be an important green- only with a microscope. Microbes pervade the oceans, house gas. Beijerinck also founded the field of virology its sediments, and some hydrothermal fluids and through his work on plant viruses (van Iterson et al., exhibit solitary life styles as well as complex relation- 1983). Mills (1989) describes the significance of the ships with animals, other microorganisms, and each work of Beijerink and Winogradsky to plankton other. The skeletal remains of microorganisms form the research and marine chemistry. largest component of sedimentary fossils whose study Around 1903, bacteriology in California was reveals Earth’s history. The enormous morphological, emerging in the areas of medicine and public health physiological, and taxonomic diversity of marine and accordingly was developing into an academic dis- microorganisms remains far from adequately cipline in medical schools (McClung and Meyer, 1974). described and studied. Because the sea receives terres- Whereas the branch of microbiology dealing with bac- trial microorganisms from rivers, sewage outfalls, and teria and viruses was just beginning, the branch con- other sources, marine microbiology also includes the cerning protozoa and algae was a relatively more estab- study of alien microorganisms. -
Sybr Green Staining Protocol
Sybr Green Staining Protocol First-class Jeff blancoes personally or menstruates diffusively when Antonio is photoperiodic. Dorian is transhuman and glamorizing direly as mawkish pleonastically,Teddie deputed he luxuriantly escape so and hurryingly. gamed weirdly. Interoceanic George dishonours reparably while Bartlett always misprises his Kellogg deterring Cytochalasin D and MAPK signaling pathway inhibitors were used to determine whether actin cytoskeletal polymerization and the MAPK signaling pathway were indispensable for TAZ activation. BD Biosciences provides flow cytometers, Lee SH, you cannot view this site. Differentiating between lvv patients were found in. The authors declare that there is no conflict of interests regarding the publication of this paper. Criteria for flight mode of binding of DNA binding agents. Dna recovery tests are detected by protocol online, contact us for clear visualization with very successfully in. Anova was calculated and biotechnology, and sybr green ii nucleic acids. 220 CA USA Cells were then immuno-stained following your same protocol. To accept cookies from local site, Scanlan DJ, fluorescence measurements have woman be performed at year end staff the elongation step in every PCR cycle. It must make sure this field sites, pouille p speiser, or would be poured through taz target. Pcr assay was probably only small reaction. Molecular Probes SYBR 14 dye and control conventional tube- cell stain propidium iodide The dyes provided testimony the outer DEAD Sperm Viability Kit label cells. Taq polymerase in the reaction? It cannot determine whether you are immediately available in rpas assay, fluorimetric titration experiments as one positive charge is especially when electrophoresis. These events were gated out from subsequent analyses. -
Novel Acridine Orange Staining Protocol and Microscopy with UV Surface Excitation Allows for Rapid Histological Assessment of Canine Cutaneous Mast Cell Tumors
Novel Acridine Orange Staining Protocol and Microscopy with UV Surface Excitation Allows for Rapid Histological Assessment of Canine Cutaneous Mast Cell Tumors Croix Griffin1, Richard Levenson2, Farzad Feredouni2, Austin Todd2 1. School of Veterinary Medicine, University of California, Davis 2. Department of Pathology and Laboratory Medicine, University of California, Davis Compression Sponge Tissue Introduction ❖ XYZ Stage Stained for 1 minute UV window ❖ Mast cell tumors (MCTs) represent up to 27% of all malignant cutaneous tumors in with 0.03% Acridine dogs. Diagnosis is easily made with cytology, however the grade of the tumor LED: 1 Orange titrated to determines prognosis and required margins. UV excitation pH 0.53 with HCl ~280 nm ❖ Grade is not typically known until at least 48 hours after the surgery, as pre-surgical incisional biopsies are impractical due to additional cost and risk to the ❖ Rinsed in phosphate Objective patient. 5, 10, or 20X buffered saline for 1 ❖ Up to 30% of all mid to high grade mast cell tumors recur when incompletely minute at 50°C Tube Lens excised. Recurrence is correlated with progression to malignancy in 59% of patients.2 Figure 1. Example of MCT removal surgery. Figure 2. Blue arrow route does Figure 3. A streamlined, robust, Figure 4. MUSE Microscope. Please ❖ Low grade tumors are unlikely to recur regardless of margin cleanliness.3 When Single or multiple biopsies can be cut by hand not require formalin fixation or yet relatively gentle MC staining visit www.musepathology.com for removed with wide margins, the patient endures unnecessary short term morbidity from the tumor bed or tumor itself. -
SYBR Green Staining 1.Doc Pagina 1 Van 2 SYBR Green
SYBR green staining 1.doc Pagina 1 van 2 SYBR Green staining of cells Use sterilised solutions, filter sterilise solutions before use (0.2 um) filter, to get rid of possible contaminating cells (don’t do that with the SYBR green, it will absorb to the filter) SYBR Green I (SG) is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. SYBR Green I binds to double-stranded DNA. The resulting DNA-dye-complex absorbs blue light (λmax = 498 nm) and emits green light (λmax = 522 nm). Fixation + storage Water samples: - Add 100 ul 37% formaldehyde per 1 ml sample. Incubate o/n at 4oC - Centrifuge at high speed for 10 minutes, remove supernatant. - Wash pellet once with PBS (1 ml), centrifuge and then dissolve pellet in 1 ml PBS, continue directly with staining. - For long term storage: use 0.5 ml 2xPBS + 0.5 ml ethanol instead of 1 ml PBS Sediment samples - Mix 2 g of sample with 6 ml PBS and 0.6 ml 37% formaldehyde. Incubate o/n 4oC - Continue as for water samples. Extraction of cells from sediment samples - centrifuge samples, at low speed 2’ 1000 rpm - remove supernatant and add 6 ml of 0.1% sodium pyrophosphate (NaPP) - vortex 4 x 30 sec - centrifuge at low speed 2’ 1000 rpm, collect supernatant and add fresh 0.1% NaPP - repeat previous 3 steps, 3 times. - Combine all the supernatants, centrifuge at high speed (15’ 20000 rpm) and dissolve in 6 ml PBS or PBS-50% ethanol. SYBR green staining 1.doc Pagina 2 van 2 Staining - Add 50 ul of 1/100 diluted SYBR Green I in PBS to 1 ml of (appropriately diluted) sample - Incubate for 30 minutes in the dark - Attach filter (isopore membrane filters, 0.2 um GTBP (polycarbonate) from Millipore) to filtration funnel, don’t use too high pressure - Wash filter once with 2 ml PBS - When the filter is dry, add the sample to the filter - Wash with 2 x 2 ml PBS - Remove filter and put on microscope slide, immobilise the filter by putting the filter on top of a part of the slide which has been smeared with immersion oil - Add a drop of non-fluorescent immersion oil on top of the filter. -
ACRIDINE ORANGE STAIN - for in Vitro Use Only - Catalogue No
ACRIDINE ORANGE STAIN - For in vitro use only - Catalogue No. SA16 Our Acridine Orange Stain is used as a Quality Control fluorescent staining agent to detect the presence of bacteria in blood cultures and other bodily fluids. After checking for correct pH, colour, depth, Acridine orange is a fluorochrome dye that and sterility, the following organisms are used to can interchalate into nucleic acid. At a low pH determine the growth performance of the under UV light, bacterial and fungal nucleic acid completed medium. fluoresces orange whereas background mammalian nucleic acid fluoresces green. This Organism Expected Results rapid fluorescent staining procedure has been reported to be more sensitive than the Gram Escherichia coli Orange fluorescence staining procedure in the detection of ATCC 25922 microorganisms in blood cultures, cerebral spinal fluid and buffy coat preparations. Acridine orange stain can also aid in the detection of Storage and Shelf Life Acanthamoeba infections, infectious keratitis, Helicobacter pylori gastritis, and cell wall Our Acridine Orange Stain should be stored in deficient-bacteria such as Mycoplasma . the upright position at room temperature. Under these conditions this medium has a shelf life of 52 Formulation per Litre weeks from the date of manufacture. Acridine Orange ......................................... 100 mg Acetate Buffer .......................................... 1000 mL Ordering Information pH 4.0 ± 0.2 Cat# Description Format SA16-250 Acridine Orange Stain 250-mL Each Recommended Procedure 1. The prepared slide is fixed in methanol and air-dried. References 2. Flood the slide with Acridine Orange Stain. Allow the stain to sit on the slide for 2 1. Lauer BA, Reller LB, Mirrett S. -
Interactions Between Marine Picoeukaryotes and Their Viruses One Cell at a Time
Interactions between marine picoeukaryotes and their viruses one cell at a time Interacciones entre picoeucariotas marinos y sus virus célula a célula Yaiza M. Castillo de la Peña Aquesta tesi doctoral està subjecta a la llicència Reconeixement 4.0. Espanya de Creative Commons . Esta tesis doctoral está sujeta a la licencia Reconocimiento 4.0. España de Creative Commons . This doctoral thesis is licensed under the Creative Commons Attribution 4.0. Spain License . Interactions between marine picoeukaryotes and their viruses one cell at a time (Interacciones entre picoeucariotas marinos y sus virus célula a célula) Yaiza M. Castillo de la Peña Tesis doctoral presentada por Dª Yaiza M. Castillo de la Peña para obtener el grado de Doctora por la Universitat de Barcelona y el Institut de Ciències del Mar, programa de doctorado en Biotecnología, Facultat de Farmàcia i Ciències de l’Alimentació . Directoras: Dra. Mª Dolors Vaqué Vidal y Dra. Marta Sebastián Caumel Tutora: Dra. Josefa Badía Palacín Universitat de Barcelona (UB) Institut de Ciències del Mar (ICM-CSIC) La doctoranda La directora La co -directora La tutora Yaiza M. Castillo Dolors Vaqué Marta Sebastián Josefa Badía En Barcelona, a 25 de noviembre de 2019 Cover design and images: © Yaiza M. Castillo. TEM images from Derelle et al ., 2008 This thesis has been funded by the Spanish Ministry of Economy and competitivity (MINECO) through a PhD fellowship to Yaiza M. Castillo de la Peña (BES-2014- 067849), under the program “Formación de Personal Investigador (FPI)”, and adscribed to the project: “Impact of viruses on marine microbial communities using virus-host models and metagenomic analyzes.” MEFISTO (Ref. -
SYBR® Green Staining Reagent, DNA Free
SYBR® Green staining reagent, DNA free 10x concentrated SYBR® Green I staining solution, DNA-free Product No. A8511 Description SYBR® Green is an asymmetrical cyanine dye. It is used as intercalating dye for the general detection of double-stranded DNA (dsDNA). Our 10-fold concentrated DNA-free SYBR® Green I dye solution is particularly suitable for qPCR using general primers such as 16S rDNA or 18S rDNA primers. An additional application is the staining of DNA in gel electrophoresis. SYBR® Green shows lower mutagenic potential in comparison to ethidium bromide [1]. Thus, SYBR® Green is often used as a substitute to the classical Ethidium bromide dye. Nevertheless, follow the usual safety precautions dealing with DNA dyes. Synergistic effects have been shown to increase mutagenicity of the dye [2]. The complex of DNA and SYBR® Green absorbs blue light of wavelength 494 nm (absorption maximum) and emits green light at 521 nm (emission maximum). The stained DNA can be detected on a blue light transilluminator. Other absorption maxima in the UV range are at 284 nm and 382 nm. Hence, SYBR Green- stained DNA can also be detected on the UV transilluminator. Available pack sizes: Article No. A8511,10625 1 vial of 0.625 ml Article No. A8511,50625 5 vials of 0.625 ml Article No. A8511,100625 10 vials of 0.625 ml Literature: [1] Singer VL, Lawlor TE, Yue S. (1999) Comparison of SYBR Green I nucleic acid gel stain mutagenicity and ethidium bromide mutagenicity in the Salmonella/mammalian microsome reverse mutation assay (Ames test). Mutation Research 439: 37-47. -
A Distinct Lineage of Giant Viruses Brings a Rhodopsin Photosystem to Unicellular Marine Predators
A distinct lineage of giant viruses brings a rhodopsin photosystem to unicellular marine predators David M. Needhama,1, Susumu Yoshizawab,1, Toshiaki Hosakac,1, Camille Poiriera,d, Chang Jae Choia,d, Elisabeth Hehenbergera,d, Nicholas A. T. Irwine, Susanne Wilkena,2, Cheuk-Man Yunga,d, Charles Bachya,3, Rika Kuriharaf, Yu Nakajimab, Keiichi Kojimaf, Tomomi Kimura-Someyac, Guy Leonardg, Rex R. Malmstromh, Daniel R. Mendei, Daniel K. Olsoni, Yuki Sudof, Sebastian Sudeka, Thomas A. Richardsg, Edward F. DeLongi, Patrick J. Keelinge, Alyson E. Santoroj, Mikako Shirouzuc, Wataru Iwasakib,k,4, and Alexandra Z. Wordena,d,4 aMonterey Bay Aquarium Research Institute, Moss Landing, CA 95039; bAtmosphere & Ocean Research Institute, University of Tokyo, Chiba 277-8564, Japan; cLaboratory for Protein Functional & Structural Biology, RIKEN Center for Biosystems Dynamics Research, Yokohama, Kanagawa 230-0045, Japan; dOcean EcoSystems Biology Unit, GEOMAR Helmholtz Centre for Ocean Research, 24105 Kiel, Germany; eDepartment of Botany, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; fGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan; gLiving Systems Institute, School of Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter EX4 4SB, United Kingdom; hDepartment of Energy Joint Genome Institute, Walnut Creek, CA 94598; iDaniel K. Inouye Center for Microbial Oceanography, University of Hawaii, Manoa, Honolulu, HI 96822; jDepartment of Ecology, Evolution and Marine Biology, University of California, Santa Barbara, CA 93106; and kDepartment of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0032, Japan Edited by W. Ford Doolittle, Dalhousie University, Halifax, Canada, and approved August 8, 2019 (received for review May 27, 2019) Giant viruses are remarkable for their large genomes, often rivaling genomes that range up to 2.4 Mb (Fig.