1. Configuração do Instrumento <ol><li> Ligue o espectrofluorímetro e deixe aquecer por 1 hora. </li><li> Usando o Excel cria um arquivo com as Normas e os dados da amostra, como mostrado na Figura 1 e salve-o em comma separated values ​​&quot;csv&quot; formato. <img src="/files/ftp_upload/2465/2465fig1.jpg" alt="Figura 1" /> Figura 1. Padrões e dados de amostras. </li><li> Configurar fluorímetro da seguinte forma: <ol><li> Clique na <img src="/files/ftp_upload/2465/2465icon1.jpg" alt="Ícone de uma" /> botão, isto irá abrir a janela de Método de Análise, mostrado na Figura 2. <img src="/files/ftp_upload/2465/2465fig2.jpg" alt="Figura 2" /> Figura 2. Janela Método de Análise, na guia Geral. Faça as seguintes seleções: <ul><li> Na &quot;medição&quot; de campo, selecione Fotometria do menu suspenso. </li><li> Em &quot;Operador&quot;, digite o nome do operador apropriado. </li><li> Não há necessidade de inserir informações no &quot;Instrumento&quot; campo. Esta é selecionado automaticamente pelo software. </li><li> A &quot;amostragem&quot; de campo é inativa quando se utiliza o leitor de microplacas. </li><li> Digite comentários que você pode querer incluir no relatório no campo &quot;Comentários&quot;. </li><li> No &quot;acessório&quot; de campo, insira as informações relacionadas com os acessórios utilizados para este teste. </li><li> Nota os botões do lado direito da janela. </li><li> O botão &quot;Load&quot; permite carregar Métodos de Análise salvo anteriormente. </li><li> O botão &quot;Salvar&quot; permite a atualização de um conjunto de parâmetros de teste ou método de análise anteriormente salvo e carregado, usando o mesmo nome. </li><li> O &quot;Salvar como&quot; botão permite salvar um novo conjunto de parâmetros de teste ou Método de Análise com um nome desejado. </li></ul> Agora clique em &quot;quantificação&quot; guia, veja a Figura 3 e faça as seguintes seleções. <img src="/files/ftp_upload/2465/2465fig3.jpg" alt="Figura 3" /> Figura 3. Janela Método de Análise, guia quantitativa. <ul><li> No &quot;A determinação quantitativa do tipo&quot; campo, selecione Wavelength. Isto indica a leitura de fluorescência será feita utilizando o comprimento de onda selecionado. </li><li> Na &quot;calibragem&quot; tipo de campo, selecione 1 ª ordem. </li><li> No &quot;Número de comprimentos de onda&quot; de campo, selecione 1. </li><li> Na &quot;Concentração unidade&quot; de campo, entrar ng / mL. </li><li> Deixe desmarcado as caixas para &quot;Manual de Calibração&quot; e &quot;Força através da curva de zero&quot;. </li><li> No &quot;Digit após ponto decimal&quot; campo, selecione 2. </li><li> Na &quot;Baixa-limite de concentração&quot;, digite 0. </li><li> No campo limite &quot;superior de concentração, digite 10000. </li></ul> Agora clique em &quot;Instrumento&quot; guia e faça as seguintes seleções. <img src="/files/ftp_upload/2465/2465fig4.jpg" alt="Figura 4" /> Figura 4. Janela de análise do método, guia Instrumento <ul><li> No &quot;modo de dados:&quot; campo, selecione Fluorescência. </li><li> O &quot;Chopping velocidade&quot; campo está inativo no momento, ele é usado somente para medições de fosforescência. </li><li> No &quot;modo de comprimento de onda&quot; de campo, selecione Ambos fixo WL. </li><li> Na &quot;EX / WL1&quot; field entrar 480nm. </li><li> No &quot;EM / WL1&quot; field entrar 520nm. </li><li> Todos os outros campos sob EX e EM estão inativos no momento. </li><li> Na &quot;EX fenda&quot; campo selecione 5.0nm do menu suspenso. </li><li> No &quot;EM fenda&quot; campo selecione 5.0nm do menu suspenso. </li><li> Deixe desmarcada a caixa na frente da &quot;Tensão PMT 1-1000V&quot; campo. </li><li> No &quot;PMT Voltage&quot; campo, selecione 700V do menu suspenso. </li><li> No &quot;Auto calc estatística. Número&quot; campo selecione 2. </li><li> No &quot;Replica&quot; campo selecione 1. </li><li> No &quot;tempo de integração&quot; campo selecionar 0.1s. &lt;/ Li&gt; <li> No &quot;Delay&quot; campo de digitar ou selecionar 0s. </li></ul> Clique no separador &quot;Monitor&quot;. <img src="/files/ftp_upload/2465/2465fig5.jpg" alt="Figura 5" /> Figura 5. Janela de análise do método, guia Monitor. <ul><li> No &quot;eixo Y&quot;, &quot;Max:&quot; campo, selecione 10000 </li><li> No &quot;eixo Y&quot;, &quot;Min:&quot; campo, selecione 0. </li><li> Selecione a caixa à esquerda do &quot;Aberto de processamento de dados após a aquisição de dados&quot;. </li><li> Deixe desmarcada a caixa à esquerda do &quot;relatório de impressão depois de aquisição de dados&quot;, a menos que você queira um relatório a ser impresso automaticamente após as leituras sejam concluídas. </li></ul> Clique no botão &quot;Report&quot; guia, veja a Figura 6. <img src="/files/ftp_upload/2465/2465fig6.jpg" alt="Figura 6" /> Figura 6. Janela de análise do método, guia do relatório. <ul><li> Na &quot;saída&quot; de campo, selecionar Imprimir relatório do menu suspenso. Você também pode selecionar &quot;Use Microsoft Excel&quot; se você deseja que os dados exportados para o Excel, ou &quot;Use Folha Generator Imprimir&quot; se estiver usando o suplemento opcional no Gerador de Relatórios programa que permitirá gerar relatórios personalizados feitos. </li><li> Clique nas caixas para os itens que você quer ser incluído no relatório. </li><li> Para salvar todos os parâmetros selecionados nas diferentes guias do &quot;Método de Análise&quot; janela, clique novamente na guia &quot;Geral&quot; e salvá-las sob um nome de arquivo e em um local onde você pode encontrá-los para uso futuro, e clique no botão &quot; OK &quot;para fechar esta janela. </li><li> Isso completa a criação de parâmetros de teste do instrumento. </li><li> Agora vamos configurar o leitor de microplaca. </li><li> Clique na <img src="/files/ftp_upload/2465/2465icon2.jpg" alt="Ícone 2" /> botão, que trará a janela de configuração do MPR em cima. </li></ul> Selecione poços para os Padrões e amostras na guia Configurar MPR Window / Plate, como mostrado na Figura 7. <img src="/files/ftp_upload/2465/2465fig7.jpg" alt="Figura 7" /> Figura 7. MPR janela de instalação, guia Plate. <ul><li> Clique na posição A1 e arraste o mouse para E1, em seguida, clique na <img src="/files/ftp_upload/2465/2465icon3.jpg" alt="Ícone 3" /> botão. Isso irá selecionar as posições a serem utilizados para os padrões. Essas posições será destaque na cor verde. </li><li> Agora, precisamos selecionar as posições a F1 H3 para as amostras. Clique e arraste o mouse a partir de F1 posição, a posição H3 e depois clique no <img src="/files/ftp_upload/2465/2465icon4.jpg" alt="Ícone 4" /> botão, isto irá destacar as posições na cor amarela. </li><li> Em seguida, repita a mesma operação para os cargos de A2 E3, para selecionar as posições para medições da amostra. </li><li> Agora, vamos carregar o arquivo Comma Separated arquivo (csv) Variável preparado com antecedência com as Normas e informação de amostra. </li></ul> Clique no botão &quot;Bem informações&quot; guia da &quot;janela de configuração do MPR&quot;, como mostrado na Figura 8. <img src="/files/ftp_upload/2465/2465fig8.jpg" alt="Figura 8" /> Figura 8. MPR janela de instalação, guia de informação bem. <ul><li> Em seguida, clique na <img src="/files/ftp_upload/2465/2465icon5.jpg" alt="Ícone 5" /> botão. Uma janela do Windows Explorer será aberto que, como mostrado na Figura 9, o que permitirá localizar o &quot;Bem information.csv&quot; arquivo e carregá-lo. <img src="/files/ftp_upload/2465/2465fig9.jpg" alt="Figura 9" /> Figura 9. Carregando informações de amostra bem. Depois de carregar o &quot;Bem informações&quot; arquivo, a &quot;informação Bem&quot; vai olhar como mostrado na Figura 10 <img src="/files/ftp_upload/2465/2465fig10.jpg" alt="Figura 10" /> Figura 10. Carregado de informações de amostra bem. </li></ul></li></ol></li></ol> 2. Picogreen Preparação de amostras de Ensaio <ol><li> Prepare 1X tampão TE (10 mM Tris-HCl, 1 mM EDTA), adicionando 1 mL de 20 X tampão TEa 19 mL dI H 2 O. </li><li> Prepare PicoGreen Solução Reagente pela adição de 50 mL 200 X Reagente PicoGreen para 9,95 1 X mL tampão TE. </li><li> Elaborar normas (Lambda dsDNA) fazendo diluições em série de ações dsDNA (100 mcg / mL). Primeiro preparar uma solução de 50 mg / mL dsDNA adicionando 25 mL de estoque dsDNA (100 mcg / mL) e 25 mL 1 x tampão TE. Em seguida, preparar as soluções padrão, adicionando os volumes indicados de dsDNA e buffer como listadas na tabela abaixo: <table border="1"><tbody><tr><td> Concentração de dsDNA </td><td> Volume de dsDNA </td><td> Volume de Tampão </td></tr><tr><td> 1 mg / mL </td><td> 20 mL de 50 mcg / mL dsDNA </td><td> 980 mL 1 X tampão TE </td></tr><tr><td> 0,1 g / mL </td><td> 100 L de 1 mg / mL dsDNA </td><td> 900 mL 1 X tampão TE </td></tr><tr><td> 0,01 mcg / mL </td><td> 100 L de 0,1 mcg / mL dsDNA </td><td> 900 mL 1 X tampão TE </td></tr><tr><td> 0,001 mg / mL </td><td> 100 L de 0,01 mcg / mL dsDNA </td><td> 900 mL 1 X tampão TE </td></tr></tbody></table></li></ol> 3. Medição de Padrões e fluorescência da amostra <ol><li> Pipetar 150 mL de cada padrão em poços A1-E1 de uma placa de 96 poços, por Figura 7, e de acordo com a tabela a seguir: <table border="1"><tbody><tr><td> Bem </td><td> Padrão </td></tr><tr><td> A1 </td><td> 1 mg / mL </td></tr><tr><td> B1 </td><td> 0,1 g / mL </td></tr><tr><td> C1 </td><td> 0,01 mcg / mL </td></tr><tr><td> D1 </td><td> 0,001 mg / mL </td></tr><tr><td> E1 </td><td> 1 X tampão TE </td></tr></tbody></table></li><li> Pipetar 150 mL amostras desconhecidas em poços de F1 H3, mostrado na Figura 7 </li><li> Despeje PicoGreen solução preparada na etapa 1,2 em uma calha projetado para multi-canal de uso da pipeta. Use uma pipeta multi-canal para fornecer solução de 150 mL PicoGreen aos poços A1-H3. Solução de misturar e padrões cuidadosamente com pipeta. </li><li> Colocar a placa na área escura e esperar 2-5 minutos para a reação para se desenvolver. </li></ol> 4. Resultados representante A curva de calibração bom é avaliado utilizando os diferentes parâmetros fornecidos pelo software F-7000 em cima da medida das normas e cálculo da curva de calibração pelo software. O coeficiente de determinação indica a qualidade do ajuste dos padrões de medida e calculada a curva de calibração. Quanto mais próximo este valor é &quot;1&quot;, melhor o ajuste do valor medido e curva de calibração. Se o valor está longe de &quot;1&quot;, as normas devem ser reavaliados, preparado novamente, ou a adequação da curva de calibração mudou. A Figura 11 mostra um exemplo de uma curva de calibração com um coeficiente de determinação calculado = 0,99993, obtidos para a quantificação de dsDNA usando PicoGreen corante. <img src="/files/ftp_upload/2465/2465fig11.jpg" alt="Figura 11" /> Figura 11. Exemplo de dsDNA curva de calibração.

<ol>
	<li>Singer, V. L., Jones, L. J., Yue, S. T., Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+PicoGreen+reagent+and+development+of+a+fluorescence-based+solution+assay+for+double-stranded+DNA+quantitation.">Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation.</a> Analytical Biochemistry. 249 (2), 228-238 (1997).</li></ol>

Luis A. Moreno e Kendra L. Cox são empregados por Hitachi High Technologies Latina que produz o instrumento utilizado neste artigo.

Pipetagem cuidadosa durante a preparação da amostra, bem como a utilização de um espectrofotômetro de fluorescência estável e sensível é essencial para obter bons resultados e reprodutíveis. No caso do espectrofotômetro de fluorescência utilizado para este teste, sugere-se com 300 mL de amostra de volume final no leitor de microplacas. Menor volume diminui a sensibilidade, e um volume maior pode causar contaminação cruzada entre os poços.

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<td>Distilled H2O</td>
<td>Reagent</td>
<td>&nbsp;</td>
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<td>Quant-iT Picogreen dsDNA Assay Kit from Invitrogen</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>P7589</td>
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<td>Nalge Nunc Fluorescence Microplates p/n 237108</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>237108</td>
<td>&nbsp;</td>
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<td>12 Channel Eppendorf Micropipette</td>
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<td>&nbsp;</td>
<td>3516677</td>
<td>&nbsp;</td>
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<td>1000 &mu;L Eppendorf Pipette</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>200 &mu;L Eppendorf Pipette </td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>10 mL serological pipet</td>
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<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Hitachi F-7000 Fluorescence Spectrophotometer with FL Solutions 2.1</td>
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<td>&nbsp;</td>
<td>5J1-0003</td>
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<td>Microplate Reader Accessory for F-7000</td>
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quantification of dsdna using the hitachi f-7000 fluorescence spectrophotometer and picogreen dye

Quantificação de DNA, especialmente em pequenas concentrações, é uma tarefa importante, com uma ampla gama de aplicações biológicas, incluindo padrão ensaios de biologia molecular, tais como síntese e purificação do DNA, aplicações de diagnóstico, tais como a quantificação de produtos de amplificação de DNA, e detecção de moléculas de DNA em drogas preparações. Durante este vídeo vamos demonstrar a capacidade do espectrofotômetro Hitachi F-7000 de fluorescência equipado com um acessório Placa Micro Reader para realizar dsDNA quantificação usando Molecular Probes Quant-it PicoGreen kit reagente corante. O Espectrofotômetro de Fluorescência F-7000 oferece alta sensibilidade e medições em alta velocidade. É um sistema altamente flexível, capaz de medir a fluorescência, luminescência, e fosforescência. Vários modos de medição estão disponíveis, incluindo comprimento de onda de digitalização, digitalizar tempo, fotometria e 3-D de medição de varredura. O espectrofotômetro tem sensibilidade na faixa de 50 picomoles de fluoresceína quando se utiliza um volume de amostra 300 mL na microplaca, e é capaz de medir velocidades de digitalização de 60.000 nm / minuto. Ele também tem uma ampla faixa dinâmica de até 5 ordens de magnitude, que permite o uso de curvas de calibração em uma ampla faixa de concentrações. O sistema óptico utiliza todos óptica reflexiva para o máximo de energia e sensibilidade. A faixa de comprimento de onda padrão é de 200 a 750 nm, e pode ser estendido a 900 nm quando se usa um dos opcionais perto fotomultiplicadores infravermelho. O sistema permite o controle de temperatura opcional para o leitor de placas 5-60 graus Celsius usando um externo opcional de temperatura controlada circulador de líquido. O leitor de microplaca permite o uso de 96 microplacas bem, ea velocidade de medição para 96 ​​poços é menos de 60 segundos ao usar o modo cinética. Controles de software para a F-7000 e Leitor de Microplacas também são altamente flexíveis. As amostras podem ser definidos em formatos uma coluna ou linha, e qualquer combinação de poços podem ser escolhidas para medições da amostra. Isto permite uma utilização óptima da microplaca. Além disso, o software permite importar as configurações da placa micro amostra criados no Excel e salvas em valores separados por vírgula, ou &quot;csv&quot; formato. Configurações microplaca de medição podem ser salvas e recuperadas pelo software para a conveniência e aumento da produtividade. Resultados dos dados pode ser saída para um relatório padrão, para o Excel, ou para um programa opcional Report Generator.

Watch this Scientific Journal Video about Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye at JoVE.com

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Demonstração de quantificação de dsDNA usando Molecular Probes PicoGreen corante e Hitachi Espectrofotômetro de Fluorescência F-7000 equipado com um acessório de leitor de microplacas.

Quantificação de dsDNA usando a Hitachi F-7000 Espectrofotômetro de Fluorescência e PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

quantification-dsdna-using-hitachi-f-7000-fluorescence

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Biology

24.4K visualizações.  Hitachi High Technologies America. Demonstração de quantificação de dsDNA usando Molecular Probes PicoGreen corante e Hitachi Espectrofotômetro de Fluorescência F-7000 equipado com um acessório de leitor de microplacas.

Vídeo: Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 &mu;L. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

Microvolume determinação da concentração de proteína utilizando o NanoDrop 2000c Espectrofotômetro

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

Biologia

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Lapso de tempo de imagens de Mitose Depois de Transfection siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Quantificação de γH2AX Focos em resposta à radiação ionizante

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

Determinação de concentração de ácidos nucléicos e proteínas Usando o Micro-volume Bio-spec Espectrofotômetro Nano

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Medição Rendimento Absoluto Quantum of amostras de pó

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

Visualização de Caenorhabditis elegans Estruturas cutícula Usando o Dye lipofílicos Vital, DII

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualização e análise de moléculas de mRNA Usando Fluorescência<em&gt; In Situ</em&gt; A hibridação em<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

Observação e Quantificação de Telomere e Sequências Repetitivas Utilizando Fluorescência<em&gt; In Situ</em&gt; Fluorescente (FISH) com PNA Sondas em<em&gt; Caenorhabditis elegans</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods are essential to provide critical insights into these thermogenic adipose depots. Here, the protocol presents a PET/MR imaging-based method to evaluate the activity of brown and beige adipocytes in mouse interscapular brown adipose tissue (iBAT) and inguinal subcutaneous white adipose tissue (iWAT). Visualization and quantification of the thermogenic adipose depots were achieved using [18F]FDG, the non-metabolizable glucose analog, as the radiotracer, when combined with the precise anatomical information provided by MR imaging. The PET/MR imaging was conducted 7 days after cold acclimation and quantitation of [18F]FDG signal in different adipose depots was conducted to assess the relative mobilization of thermogenic adipose tissues. Removal of iBAT substantially increased cold-evoked [18F]FDG uptake in iWAT of the mice.

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.

Visualização e Quantificação de Tecidos Adiposos Marrons e Bege em Camundongos usando [18F]FDG Micro-PET/MR Imaging

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can be used to visualize and quantify competitive interactions between different bacterial strains at the single-cell level. The protocol described here provides methods for advanced approaches in slide preparation on both upright and inverted epifluorescence microscopes, live-cell and time-lapse imaging techniques, and quantitative image analysis using the open-source software FIJI. The approach in this manuscript outlines the quantification of competitive interactions between symbiotic Vibrio fischeri populations by measuring the change in area over time for two coincubated strains that are expressing different fluorescent proteins from stable plasmids. Alternative methods are described for optimizing this protocol in bacterial model systems that require different growth conditions. Although the assay described here uses conditions optimized for V. fischeri, this approach is highly reproducible and can easily be adapted to study competition among culturable isolates from diverse microbiomes.

This manuscript describes a method for using single-cell fluorescence microscopy to visualize and quantify bacterial competition in coculture.

Quantificação da Competição Interbacteriana usando imagens de fluorescência unicelular

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...