DOCSLIB.ORG

Overcoming Cloning Problems by Staining Agarose Gels with Crystal Violet Instead of Ethidium Bromide in Lactate Dehydrogenase Ge

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Overcoming Cloning Problems by Staining Agarose Gels with Crystal Violet Instead of Ethidium Bromide in Lactate Dehydrogenase Ge Acta Biologica Hungarica 56 (3–4), pp. 389–397 (2005) OVERCOMING CLONING PROBLEMS BY STAINING AGAROSE GELS WITH CRYSTAL VIOLET INSTEAD OF ETHIDIUM BROMIDE IN LACTATE DEHYDROGENASE GENE FROM PLASMODIUM VIVAX AND PLASMODIUM FALCIPARUM D. TURGUT-BALIK,1* V. ÇELIK,1 KATH MORETON2 and R. L. BRADY2 1 Department of Biology, Faculty of Arts and Sciences, University of F°rat, Elaz°=, Turkey 2 Department of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, U.K. (Received: August 9, 2004; accepted: November 9, 2004) In this study, lactate dehydrogenase gene from Plasmodium vivax has been tried to subclone into an expression vector. Some of the Plasmodium falciparum lactate dehydrogenase mutant genes have also been tried to clone and subclone into a vector, but we failed to clone or subclone either of the genes. DNA visualisation in electrophoretic gels typically requires UV radiation and the fluorecent dye ethidium bro- mide. A crystal violet-stained gel was run instead of an ethidium bromide gel and so avoided the use of UV radiation. This enabled us to clone or subclone both Plasmodium vivax lactate dehydrogenase gene and Plasmodium falciparum lactate dehydrogenase mutant genes into any desired vector. Keywords: Plasmodium vivax – Plasmodium falciparum – lactate dehydrogenase – crystal violet – ethid- ium bromide INTRODUCTION Drug resistance of Plasmodium to currently available antimalarials is increasing throughout the world. Discovery of a new, effective, safe, permanent and inexpen- sive antimalarial is essential to overcome this problem. The enzyme lactate dehydrogenase (LDH) has been targeted for the design of a novel antimalarial drug. It is a vital enzyme for the malarial parasite. The parasite lives in the red blood cells by converting glucose to lactate using this enzyme. Because malaria parasite depends on this metabolic pathway [2, 17], compounds which block LDH of the parasite, but not of the human, also kill the plasmodium in whole blood [10]. Plasmodium falciparum LDH (PfLDH) gene was cloned from K1 and PF FCBR strains [13], related protein over-produced [14] and enzyme’s ternary structure deter- mined [4]. All these studies showed that, unlike human M4-LDH, PfLDH has a cleft on the enzyme’s surface which may be an attractive target for inhibitor design. The same study is aimed to apply to Plasmodium vivax (Pv) which is the most common * Corresponding author; e-mail: [email protected] Abbreviations: LDH: L-lactate dehydrogenase; PvLDH: lactate dehydrogenase from Plasmodium vivax 0236-5383/$ 20.00 © 2005 Akadémiai Kiadó, Budapest 390 D. TURGUT-BALIK et al. species in the world. PvLDH was succesfully cloned into the vector pGEM-T, then subcloned into the vector pKK223-3 and active protein expressed in E. coli [15]. But subcloning of the PvLDH gene and also PfLDH mutant genes was problematic. We cloned [13] and subcloned the PfLDH gene from K1 and PF FCBR strains without any problem [14]. But it was not possible for us to subclone the PvLDH gene or clone or subclone the PfLDH mutant genes for further studies for the last 1.5 years. Many different procedures were applied for cloning or subcloning studies but we were unable to obtain any positive result until the agarose gel was stained with crystal vio- let instead of ethidium bromide when the DNA was prepared for preparative pur- poses [9, 3]. Gel electrophoresis is not only used as an analytical method, it is routinely used preparatively for the purification of specific DNA fragments. Agarose is the most common matrix used to separate DNA molecules electrophoretically. It is convenient for separating DNA fragments ranging in size from a few hundred base pairs to about 20 kb [8]. A particular advantage of gel electrophoresis is that the DNA bands can be readily detected at high sensitivity. DNA bands in the gel are stained with the intercalating dye ethidium bromide. This is a positively charged polycyclic aromatic compound,which binds to DNA by inserting itself between the base pairs [16]. As lit- tle as 0.05 μg of DNA in one band can be detected as visable fluoresence when the ethidium bromide stained gel is illuminated with ultraviolet (UV) light [8]. Although very sensitive and effective, this procedure involves potential exposure to a mutagen (ethidium bromide) and use of hazardous UV light [18]. But there are some alterna- tive techniques to stain agarose gels. It is possible to stain an agarose gel with crys- tal violet [9], which binds to DNA in a similar way to ethidium bromide [7], meth- ylene blue [19, 5], Nile blue [1] or brilliant cresyl blue [12]. Using such dyes was thought to be not as harmful as ethidium bromide and the DNA fragments can be visualised on a simple light box that is not as hazardous as UV light. Therefore, in this study, crystal violet stained agarose gels were used instead of ethidium bromide when the DNA was needed for preparative purposes. This enabled us to clone or subclone both LDH gene from Plasmodium vivax and LDH gene mutants from Plasmodium falciparum into any desired vector. In this paper, gel pic- tures are shown for PvLDH, PfLDH gene mutants and for PfLDH as control, but insertion of PvLDH gene into only expression vector pKK223-3 will be explained as an example to avoid any confusion. MATERIALS AND METHODS Bacterial strains and growth media The PvLDH was subcloned into pKK233-3 (Pharmacia Biotech, Sweden), and pro- tein expressed in Escherichia coli DH5a {supE44 ΔlacU169 (φ80 lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1}. The E. coli DH5α cells were cultured in double strength (2X) YT broth. Where appropriate ampicillin (100 μg/ml) was used in media for the selection and growth of transformants. Acta Biologica Hungarica 56, 2005 Overcoming cloning problems in Plasmodial LDHs 391 Agarose gel electrophoresis using ethidium bromide Agarose gel electrophoresis was performed in a horizontal gel apparatus. The gel was made up in 1 × TAE (0.04 M Tris-acetate, 0.001 M EDTA pH: 8.0) containing 1% agarose in a total volume of 50 ml. The solution was boiled in a microwave oven. It was cooled to hand hot before addition of 5 μl of 10 mg/ml ethidium bromide and poured into a cast and left to gel. The DNA samples containing 2 ml of sample appli- cation buffer (4 g sucrose, 0.5 ml 2 M tris, 2 ml 0.5 M EDTA, and 4 mg bromophe- nol blue in 10 ml dH2O) were loaded onto gel. Electrophoresis was carried out in 1 × TAE at 40–50 mA until the bromophenol blue dye front had migrated to about 2/3 the length of the gel. After electrophoresis the DNA was visualized on an ultraviolet transilluminator. Specific DNA fragments may be cut out of the gel and the DNA in the gel slices cleaned and purified using Qiagen QIAquick Gel extraction Kit (Qiagen U.K., Cat. No: 28704). Agarose gel electrophoresis using crystal-violet Agarose gel electrophoresis was performed in a horizontal gel apparatus. The gel was made up in 1 × TAE (0.04 M Tris-acetate, 0.001 M EDTA pH: 8.0) containing 1% agarose in a total volume of 50 ml. The solution was boiled in a microwave oven. It was cooled to hand hot before addition of crystal violet to the final concentration of 2 μg/ml [9, 3] and poured into a cast and left to gel. The DNA samples containing 2 μl of sample application buffer (50% glycerol, crystal violet to the final concentra- tion of 2 μg/ml in 10 μl 1 × TAE buffer) were loaded onto the gel against a white background. Electrophoresis was carried out in 250 ml of 1 × TAE containing crystal violet to the final concentration of 2 μg/ml at 40–50 mA. DNA bands were visable as they were separating during the electrophoresis. The gel was run until the blue dye was migrated to about 2/3 the length of the gel. Specific DNA fragments were cut out of the gel using a scalpel in as small a gel slice as possible and the DNA in the gel slices cleaned and purified using Qiagen QIAquick Gel extraction Kit. Restriction of DNA The DNA coding for LDH from P. vivax and the expression vector pKK223-3 were digested with the restriction enzymes EcoRI and PstI under the conditions recom- mended by the suppliers of each enzyme. The reaction was as follows: 30 μl DNA (from the gel extraction), 4 μl 10 × Buffer (supplied with enzymes), 2 μl (12 U/μl) μ μ μ μ Eco RI, 2 l (10 U/ l) Pst I and 2 l dH2O to the final volume of 40 l. Restriction digests were performed in a waterbath, at 37 °C, for 1 h. After the digest they were run out on an either crystal violet or ethidium bromide stained gel where necessary. After the electrophoresis, PvLDH DNA and vector DNA were cleaned by using Qiagen QIAquick Gel extraction Kit and their concentrations were estimated on 1% agarose gel, stained with ethidium bromide, prior to ligation. Acta Biologica Hungarica 56, 2005 392 D. TURGUT-BALIK et al. Ligation The Eco RI/Pst I digested PvLDH gene was ligated into a similarly digested expres- sion vector pKK223-3. The reaction was carried out using the enzyme T4 DNA lig- ase (Promega, USA) as follows: 100 ng DNA (insert), 4 : 1 Insert: vector ratio (molar), 2 μl 100 × Ligase buffer (supplied with enzyme), 1 μl (3 U/μl) T4 DNA lig- μ ase and dH2O to the final volume of 20 l. Ligations were set up at 4 °C for 16 h in an Eppendorf thermal cycler.
Load more

Recommended publications
  • New Nile Blue Derivatives As NIR Fluorescent Probes and Antifungal Agents †
    Proceedings New Nile Blue Derivatives as NIR Fluorescent Probes † and Antifungal Agents Rui P. C. L. Sousa 1,2, João C. C. Ferreira 1,2, Maria João M. F. Sousa 2 and M. Sameiro T. Gonçalves 1,* 1 Centre of Chemistry, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal 2 Centre of Molecular and Environmental Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal * Correspondence: [email protected] † Presented at the 22nd International Electronic Conference on Synthetic Organic Chemistry, 15 November– 15 December 2018; Available Online: https://sciforum.net/conference/ecsoc-22. Published: 14 November 2018 Abstract: The synthesis of four new Nile Blue derivatives with hydrogen, propyl and/or aminopropyl groups as substituents of the amines of 5- and 9-positions is described. Photophysical properties were evaluated in acidified ethanol and aqueous solution at physiological pH. Antifungal activity is also studied through the obtention of MIC values. Keywords: benzo[a]phenoxazines; Nile Blue derivatives; NIR fluorescent probes; antifungal agents 1. Introduction The development of new near-Infrared (NIR) fluorescent probes is a very important issue due to the wide range of applications [1–4]. These probes are an excellent choice to label biological material since its emission will not interfere with the natural fluorescence of biological compounds. Benzo[a]phenoxazinium salts, with Nile Blue being the best known, display fluorescence at around 600 nm and have been used as covalent and non-covalent fluorescent probes for amino acids, proteins and DNA, among other biological material [5–10]. In addition, applications as sensors or agents for photodynamic therapy (PDT) have been described [11,12].
    [Show full text]
  • Nile Blue Is a Basic Dye of the Oxazine Group, Which Has Been Used in Several Microscopic and Histochemical Techniques (4, 6)
    ACTA HISTOCHEM. CYTOCHEM. Vol. 16, No. 3, 1983 LETTER TO THE EDITOR NILE BLUE SULFATE STAINING FOR DEMONSTRATION OF LIPIDS IN FLUORESCENCE MICROSCOPY Nile blue is a basic dye of the oxazine group, which has been used in several microscopic and histochemical techniques (4, 6) . A staining method based on application of Nile blue sulfate was introduced early for the cytochemical demon- stration of lipids (3, 8, 9), showing neutral fats and fatty acids in red and blue, respectively. There is evidence (1, 10) that aqueous solutions of Nile blue sulfate contain the blue cation of the dye, a red oxidation product (Nile red), and the orange-red imino base. According to this view, phospholipids and fatty acids stain blue because they react with the Nile blue cation, meanwhile neutral fats appear in red color. Other observations seem to be in disagreement with this staining mecha- nism (5, 6) . A fluorescence reaction in tissues after staining with Nile blue sulfate and brilliant cresyl blue has been found by Bozzo and Campos Vidal (2). During the course of investigations by using oxazine dyes we have observed that Nile blue sulfate produces a strong fluorescence reaction in lipid droplets. The fat body of Drosophila larvae is a continuous tissue mass which has been chosen as test material because of its abundance in lipidic inclusions. Drosophila hydei testes, surrounded by the fat body, were fixed in 5 % formaldehyde for 1-24 hr, washed, and then immersed in 0.1 mg/ml aqueous Nile blue sulfate (Fluka) for 30 min. After staining, the material was briefly washed and mounted with a drop of water.
    [Show full text]
  • Dna Revealed
    Staining DNA on electrophoresis gels dna revealed Ethidium bromide, a potent mutagen Staining DNA on the move Concentrated In research laboratories, ethidium bromide and similar Recently, several commercial products have emerged that DNA Stain Dil e ute wi volum o th an equal f di use stilled water before fluorescent compounds are normally used to visualise DNA enable the DNA to be seen as it moves across the gel. Sto . re eze N at 4 °C. Do not fre AT N IO IO NA AT L C DUC ENTR GY E E FOR BIOTECHNOLO T HE U ING on a gel. Unfortunately, ethidium bromide and its breakdown Suppliers seldom reveal their composition, but several of NIVERSITY OF READ products are potent mutagens and carcinogens and therefore these stains contain Nile blue sulphate (also known as they should not be used in schools. Such dyes are often flat Nile blue A), a dye which had not previously been noted molecules with similar dimensions to DNA base pairs. When for its ability to stain DNA. Adkins and Burmeister (1996) ethidium bromide binds to DNA, it slips between adjacent give useful guidance as to its use as well as hints for identifying base pairs and stretches the double helix. This explains the other dyes which may be useful for visualising DNA. resources Methylene blue dye’s mutagenic effect — the ‘extra bases’ cause errors when the DNA replicates. In addition, short-wavelength UV light All of the dyes used for staining ‘mobile’ DNA are cationic Yung-Sharp, D. and (which itself is harmful) is required for ethidium bromide — that is, they are positively charged in the gel buffer, at Kumar, R.
    [Show full text]
  • A Consideration of the Term Gloeocystidium
    April 1976 MEMOIRS OF THE NEW YORK BOTANICAL GARDEN 28(1): 123-130 A CONSIDERATION OF THE TERM GLOEOCYSTIDIUM MICHAEL J. LARSEN AND HAROLD H. BURDSALL, JR. Center for Forest Mycology Research, Forest Products Laboratory, Forest Service, U. S. Department of Agriculture, Madison, WI 53705 Structures termed “gloeocystidia” occur in diverse genera throughout the major groups of Homobasidiomycetes and have been variously defined: Ainsworth et al. (1971) state that it is a cystidium “that is thin-walled, usually irregular and with highly refractive hyaline or yellowish contents.” Snell and Dick ( 197 1) list the variant spelling “gleocystidium” with the definition, “A special form of cystidium in Hymenomycetes, of gelatinous or horny consistency and with oily, resinous, or granular contents.” Talbot (1954) and Price (1975) have provided comprehensive statements on the concept of gloeocystidia. We present a condensed version here of Talbot’s (1954, p. 288) definition. Sterile organs, with thin walls; lack of sculpturing and encrustation; contents hyaline to brownish, highly refractive, homogeneous, granular, or oily; aris­ ing from subhymenial and contextual tissues; staining deeply in phloxine and eosine in KOH mounts and becoming brown in iodine solutions. In 1944, Romagnesi proposed the term “macrocystide” for a cystidial form in the “Lactario-russulés,” and these cystidia (macrocystidia) were described as “trés longue . fusiform ou claviforme, souvent terminée par une pointe ou un appendice variable; son pédicule est très long et souvent en connexion avec laticifères de la trame,” and secondly “très souvent, mais non toujours, devient gris-bleu ou noirâtre . au contact de la sulfovanilline. .” Romagnesi’s ( 1944) interpretation of macrocystidia is apparently based primarily on form, and secondarily on the chemical reaction with sulfovanillin.
    [Show full text]
  • Analysis of Polyhydroxyalkanoates Granules in Haloferax Mediterranei by Double-Fluorescence Staining with Nile Red and SYBR Green by Confocal Fluorescence Microscopy
    polymers Article Analysis of Polyhydroxyalkanoates Granules in Haloferax mediterranei by Double-Fluorescence Staining with Nile Red and SYBR Green by Confocal Fluorescence Microscopy Verónica Cánovas 1,2,* , Salvador Garcia-Chumillas 1,2, Fuensanta Monzó 1, Lorena Simó-Cabrera 3,4 , Carmen Fernández-Ayuso 1, Carmen Pire 3,4 and Rosa María Martínez-Espinosa 3,4,* 1 Technological Centre of Footwear and Plastic of the Region of Murcia (CETEC) Avda, Europa 4-5, 30840 Alhama de Murcia, Spain; [email protected] (S.G.-C.); [email protected] (F.M.); [email protected] (C.F.-A.) 2 Cetec Biotechnology, Avda, Europa 4-5, 30840 Alhama de Murcia, Spain 3 Department of Agrochemistry and Biochemistry, Biochemistry and Molecular Biology Division, Faculty of Science, University of Alicante, Carretera San Vicente del Raspeig s/n, San Vicente del Raspeig, 03690 Alicante, Spain; [email protected] (L.S.-C.); [email protected] (C.P.) 4 Multidisciplinary Institute for Environmental Studies “Ramón Margalef”, University of Alicante, Ap. 99, 03080 Alicante, Spain * Correspondence: [email protected] (V.C.); [email protected] (R.M.M.-E.); Tel.: +34-968-662-200 (V.C.); +34-965-903-400 (ext. 1258) (R.M.M.-E.) Abstract: Haloferax mediterranei is a haloarchaeon of high interest in biotechnology because it produces Citation: Cánovas, V.; and mobilizes intracellular polyhydroxyalkanoate (PHA) granules during growth under stress Garcia-Chumillas, S.; Monzó, F.; conditions (limitation of phosphorous in the culture media), among other interesting metabolites Simó-Cabrera, L.; Fernández-Ayuso, (enzymes, carotenoids, etc.). The capability of PHA production by microbes can be monitored with C.; Pire, C.; Martínez-Espinosa, R.M.
    [Show full text]
  • Lysosomal Localization and Mechanism of Uptake of Nile Blue Photosensitizers in Tumor Cells1
    [CANCER RESEARCH 51, 2710-2719, May 15, 1991] Lysosomal Localization and Mechanism of Uptake of Nile Blue Photosensitizers in Tumor Cells1 Chi-Wei Lin,2 Janine R. Shulok, Sandra D. Kirley, Louis Cincotta, and James W. Foley Urology Research Laboratory, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114 [C-W. L., J. R. S., S. D. KJ, and Rowland Institute for Science, Cambridge, Massachusetts 02142 ¡L,C., J. W. F.] ABSTRACT tosensitizers with high tumor selectivity will enable effective treatment of multiple, infiltratili!*, and invisible tumors, thus Nile blue derivatives have been shown to be potentially effective expanding the utility of PDT as a useful tool in cancer therapy photosensitizers for photodynamic therapy of malignant tumors. Results with intent to cure. Active research is under way to search for of a previous study suggested that the high accumulation of these dyes more tumor-selective sensitizers (4-6) and to improve the sen- in cells may be the result of dye aggregation, partition in membrane lipids, and/or sequestration in subcellular organelles. In this report, sitizer delivery system for better tumor targeting (7-10). results of studies are presented from an investigation of the subcellular Several early studies with animal tumor models have shown localization and mechanism of accumulation of these dyes in cells in that benzophenoxazines, including several Nile blue analogues, vitro. A video-enhanced fluorescence microscopy was used, and a punctate constitute a special class of dyes that are selectively localized in pattern of fluorescence was seen, most of which was localized in the tumors (11-15).
    [Show full text]
  • Handbook of Biological Dyes and Stains Synthesis and Industrial Applications
    HANDBOOK OF BIOLOGICAL DYES AND STAINS SYNTHESIS AND INDUSTRIAL APPLICATIONS R. W. SABNIS Pfizer Inc. Madison, NJ HANDBOOK OF BIOLOGICAL DYES AND STAINS HANDBOOK OF BIOLOGICAL DYES AND STAINS SYNTHESIS AND INDUSTRIAL APPLICATIONS R. W. SABNIS Pfizer Inc. Madison, NJ Copyright Ó 2010 by John Wiley & Sons, Inc. All rights reserved. Published by John Wiley & Sons, Inc., Hoboken, New Jersey Published simultaneously in Canada No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, exckpt as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization though payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 kver Street, Hoboken, NJ 07030, (201) 748-601 1, fax (201) 748-6008, or online at http://www.wiley.com/go/permission. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials.
    [Show full text]
  • Supporting Information a Nile Blue Based Infrared Fluorescent Probe
    Electronic Supplementary Material (ESI) for ChemComm. This journal is © The Royal Society of Chemistry 2014 Supporting information A Nile Blue Based Infrared Fluorescent Probe: Imaging tumors that over-express Cyclooxygenase-2 Benhua Wanga, Jiangli Fan*a, Xianwu Wangb, Hao Zhua, Jingyun Wang*b, Huiying Mua, Xiaojun Penga a State Key Laboratory of Fine Chemicals, Dalian University of Technology, No. 2 Linggong Road, High-tech District, Dalian 116024, China. b Department School of Life Science and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Ganjingzi District, Dalian 116024, China. E-mail: [email protected], [email protected]. Content Materials and methods.....................................................................................................................S2 Synthesis of Compounds.................................................................................................................S2 Scheme S1. Synthetic procedures of Niblue-C6-IMC and its intermediates.................................S2 Photophysical properties of Niblue-C6-IMC.................................................................................S3 Cell incubation and staining with Niblue-C6-IMC........................................................................S4 Native-PAGE...................................................................................................................................S4 Fluorescence image of live cell stained with NBD C6-ceramide....................................................S4
    [Show full text]
  • Nile Blue 690 Perchlorate
    PO Box 31126 Dayton, OH 45437 Tel: 937.252.2989 Fax: 937.258.3937 E-mail: [email protected] www.exciton.com NILE BLUE 690 PERCHLORATE Synonym: 5-amino-9-(diethylamino)-benzo[a]phenoxazin-7-ium perchlorate Catalog No.: 06900 CAS No.: 53340-16-2 Chemical Formula: C20H20N3O.CIO4 MW: 417.85 Appearance: Dark green crystals Structure: + (H5C2)2N O NH2 ClO - N 4 Lasing Wavelength Max. Range Pump Source Concentration Abs Fl (nm) (nm) (nm) Solvent (molar) λ-max λ-max _________________________________________________________________________________________________ 705 FL3 Methanol 624e 660m 717 689-750 FL11a Methanol 628e 722 FL Methanol 678 656-719 XeCl(308)110 Methanol 7 x 10-4 710 696-740 XeCl(308)114 Methanol 2.3 x 10-3 698 680-723 XeF(351)154 Ethanol 8 x 10-4(NB690)+ 3.8 x 10-3(R610) 681 662-710 Nd:YAG(532)58 683 Nd:YAG→CV670(647)33 Methanol 2x10-4 109 -3 698 682-715 Nd:YAG(355) MeOH/H2O,3/2 1.5 x 10 (NB690), 1.5 x 10-4(R640) 111 -4 670 657-692 N2(337) Ethanol 4 x 10 (NB690), 1.9 x 10-3(R610) 114 -3 695 683-751 N2(337) Ethanol 1.1 x 10 5 -3 696 683-710 N2(337) Ethanol 3.8 x 10 (R610), 8 x 10-4(NB690) 90 729 688-783 N2(337) Ethanol 730 692-782 Kr(cw)14 EG 750 710-790 Kr(647)17 EG 1 x 10-3 695 682-730 Cu(511,578)175 Methanol 2 x 10-3 EG = Ethylene glycol; MeOH/H2O = Methanol/water; e = ethanol; m = methanol PO Box 31126 Dayton, OH 45437 Tel: 937.252.2989 Fax: 937.258.3937 E-mail: [email protected] www.exciton.com NILE BLUE 690 PERCHLORATE REFERENCES: 3.
    [Show full text]
  • Nile Blue-Based Nano-Sized Ph Sensors for Simultaneous Far- Red and Near-Infrared Live Bioimaging
    Nile blue-based nano-sized pH sensors for simultaneous far- red and near-infrared live bioimaging Jeppe Madsen, Irene Canton, Nicholas J. Warren, Efrosyni Themistou, Adam Blanazs, Burcin Ustbas, Xiaohe Tian, Russell Pearson, Giuseppe Battaglia, Andrew L. Lewis and Steven P. Armes* 1. Table of Contents 1. Table of Contents .......................................................................................................... 1 2. Tables ............................................................................................................................ 2 3. Figures ........................................................................................................................... 2 4. Materials ........................................................................................................................ 3 5. Synthesis of NBM monomer ......................................................................................... 4 6. Synthesis of NBC monomer .......................................................................................... 4 7. Reverse-phase HPLC analysis of Nile Blue monomers ................................................ 5 8. Dye purification ............................................................................................................. 5 9. Synthesis of PMPC macro-CTA via RAFT polymerization ......................................... 5 10. ATRP of PMPC with Nile Blue dye present throughout the polymerization ............ 6 11. ATRP of MPC with Nile Blue dye added at high MPC
    [Show full text]
  • Safety Data Sheet Packet
    Date of Issue: 23 March 2016 SAFETY DATA SHEET PACKET Product Identifier SRM Number: 1934 SRM Name: Fluorescent Dyes for Quantitative Flow Cytometry (Visible Spectral Range) SRM Description: This Standard Reference Material (SRM) is intended for use in assigning fluorescence intensity values to calibration standards for quantitative flow cytometry in the visible spectral range. A unit of SRM 1934 consists of four amber ampoules, each containing 2 mL of a different fluorophore solution or suspension. The solutions include Part A: Fluorescein Solution (60 μM aqueous borate buffer solution); Part B: Nile Red Solution (60 μM acetonitrile solution); Part C: Coumarin 30 Solution (acetonitrile solution); and Part D: APC Suspension (Allophycocyanin fluorescent protein in aqueous buffer solution, 100 µL). SRM 1934 Parts: Part A: Fluorescein Solution Part B: Nile Red Solution Part C: Coumarin 30 Solution Part D: APC Suspension Company Information National Institute of Standards and Technology Standard Reference Materials Program 100 Bureau Drive, Stop 2300 Gaithersburg, Maryland 20899-2300 Telephone: 301-975-2200 Emergency Telephone ChemTrec: FAX: 301-948-3730 1-800-424-9300 (North America) E-mail: [email protected] +1-703-527-3887 (International) Website: http://www.nist.gov/srm This page intentionally left blank. DATE: 23 March 2016 Product Identifier SRM Number: 1934 SRM Name: Fluorescent Dyes for Quantitative Flow Cytometry (Visible Spectral Range) SRM Part: Part A: Fluorescein Solution Under the U.S. Department of Labor, Occupational Safety and Health Administration (OSHA) 29 CFR 1910.1200, this Standard Reference Material (SRM) is NOT classified as a physical hazard or a health hazard, a simple asphyxiant, combustible dust, pyrophoric gas, or hazard not otherwise classified.
    [Show full text]
  • Chemical Visualization of Latent Prints
    The author(s) shown below used Federal funds provided by the U.S. Department of Justice and prepared the following final report: Document Title: Chemical Visualization of Latent Prints Author: Evan Durnal Document No.: 238008 Date Received: March 2012 Award Number: 2008-NI-CX-K012 This report has not been published by the U.S. Department of Justice. To provide better customer service, NCJRS has made this Federally- funded grant final report available electronically in addition to traditional paper copies. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Chemical Visualization of Latent Prints Grant # 2008-NI-CX-K012 Final Report For National Institute of Justice Office of Justice Programs U.S. Department of Justice MRI Project No. 110636 July 30, 2010 i Preface The primary author of this report was Mr. Evan Durnal. The report was reviewed by Dr. James Egan and Mr. Tim Lanigan. This document was created in fulfillment of cooperative agreement contract # 2008-NI-CX-K012 Latent Print Chemical Visualization. This document was revised from the DRAFT final report based upon comments provided by The National Institute of Justice review panel. The period of performance for this contract was October 1, 2008 thru July 30, 2010. For technical questions or clarifications regarding this document, contact Dr. James Egan. MIDWEST RESEARCH INSTITUTE //s// Mr. Evan Durnal NIJ Technical Lead Approved: //s// Dr. James Egan NIJ Principal Investigator July 30, 2010 ii Contents Preface ................................................................................................................................. ii Tables ................................................................................................................................. iv Figures...............................................................................................................................
    [Show full text]