1. Instrumento de configuración <ol><li> Encienda el espectrofluorómetro y permitir que se caliente por 1 hora. </li><li> Uso de Excel crea un archivo con las normas y los datos de la muestra, como se muestra en la Figura 1 y guardarlo en valores separados por comas formato &quot;CSV&quot;. <img src="/files/ftp_upload/2465/2465fig1.jpg" alt="Figura 1" /> Figura 1. Las normas y los datos de las muestras. </li><li> Establecer fluorómetro de la siguiente manera: <ol><li> Haga clic en el <img src="/files/ftp_upload/2465/2465icon1.jpg" alt="Icon 1" /> botón, se abrirá la ventana Método de Análisis, que se muestra en la Fig. 2. <img src="/files/ftp_upload/2465/2465fig2.jpg" alt="Figura 2" /> Figura 2. Análisis ventana del método de la ficha General. Realice las selecciones siguientes: <ul><li> En la &quot;medición&quot;, seleccione Fotometría del menú desplegable. </li><li> En el &quot;operador&quot;, escriba el nombre del operador que sean procedentes. </li><li> No hay necesidad de introducir información en el &quot;instrumento&quot; de campo. Este es seleccionado automáticamente por el software. </li><li> El &quot;muestreo&quot; de campo está inactivo cuando se utiliza el lector de microplacas. </li><li> Introduzca los comentarios que es posible que desee incluir en el informe en el campo &quot;Comentarios&quot;. </li><li> En el &quot;accesorio&quot;, introduzca la información relacionada con los accesorios utilizados para esta prueba. </li><li> Nota de los botones en la parte derecha de la ventana. </li><li> El botón &quot;Load&quot; permite cargar previamente guardado métodos de análisis. </li><li> El botón &quot;Guardar&quot; permite la actualización de un conjunto de parámetros de prueba o de un método de análisis previamente guardado y cargado, utilizando el mismo nombre. </li><li> La opción &quot;Guardar Como&quot; botón permite guardar un nuevo conjunto de parámetros de prueba o de un método de análisis bajo el nombre que desee. </li></ul> Ahora haga clic en la &quot;cuantificación&quot; ficha, ver Figura 3 y realice las selecciones siguientes. <img src="/files/ftp_upload/2465/2465fig3.jpg" alt="Figura 3" /> Figura 3. Análisis de ventana Método, ficha cuantificación. <ul><li> En el &quot;tipo de cuantificación&quot;, seleccione la longitud de onda. Esto indica que la lectura de fluorescencia se realizará mediante la longitud de onda seleccionada. </li><li> En el &quot;tipo de calibración&quot;, seleccione una orden de st. </li><li> En el &quot;número de longitudes de onda&quot;, seleccione 1. </li><li> En la &quot;unidad de concentración&quot;, introduzca ng / mL. </li><li> Deje sin seleccionar las casillas de &quot;Calibración manual&quot; y &quot;Fuerza a través de la curva de cero&quot;. </li><li> En el &quot;dígito después del punto decimal&quot;, seleccione 2. </li><li> En el &quot;límite inferior de concentración&quot;, introduzca 0. </li><li> En el campo &quot;Límite superior de concentración, entre 10.000. </li></ul> Ahora haga clic en el &quot;Instrumento&quot; y realice los siguientes selecciones. <img src="/files/ftp_upload/2465/2465fig4.jpg" alt="Figura 4" /> Figura 4. Método de análisis ventana, pestaña Instrumento <ul><li> En el &quot;modo de datos:&quot;, seleccione fluorescencia. </li><li> La &quot;velocidad de cortar&quot; el campo está inactivo en este momento, sólo se utiliza para las mediciones de la fosforescencia. </li><li> En el &quot;modo de longitud de onda&quot;, seleccione fijos WL. </li><li> En el &quot;EX / WL1&quot; escriba 480nm. </li><li> En el &quot;EM / WL1&quot; escriba 520 nm. </li><li> Todos los demás campos de acuerdo con el antiguo y EM están inactivos en este momento. </li><li> En el &quot;corte EX&quot; de selección de campos 5.0nm del menú desplegable. </li><li> En el &quot;EM raja&quot; de selección de campos 5.0nm del menú desplegable. </li><li> Deje sin seleccionar la casilla delante de la &quot;tensión PMT 1-1000V&quot; sobre el terreno. </li><li> En el &quot;voltaje PMT&quot;, seleccione 700V desde el menú desplegable. </li><li> En el &quot;Auto Calc estadística. Número&quot; de selección de campos 2. </li><li> En la &quot;Replica&quot; de selección de campos 1. </li><li> En el &quot;Tiempo de integración&quot; de selección de campos 0.1s. &lt;/ Li&gt; <li> En el &quot;retardo&quot; escriba o seleccione 0. </li></ul> Haga clic en la ficha &quot;Monitor&quot;. <img src="/files/ftp_upload/2465/2465fig5.jpg" alt="Figura 5" /> Figura 5. Método de análisis ventana, en la ficha Monitor. <ul><li> En el &quot;eje Y&quot;, &quot;Max:&quot;, seleccione 10000 </li><li> En el &quot;eje Y&quot;, &quot;Min:&quot;, seleccione 0. </li><li> Seleccione la casilla a la izquierda de los &quot;datos abiertos de procesamiento después de la adquisición de datos&quot;. </li><li> Deje sin seleccionar la casilla a la izquierda del &quot;Informe de impresión después de la adquisición de datos&quot;, a menos que desee un informe que se imprime automáticamente después de las lecturas se han completado. </li></ul> Haga clic en el &quot;Informe&quot; ficha, vea la Figura 6. <img src="/files/ftp_upload/2465/2465fig6.jpg" alt="Figura 6" /> Figura 6. Ventana de análisis de métodos, informes de tablas. <ul><li> En la &quot;Salida&quot;, seleccione Imprimir informe del menú desplegable. También puede seleccionar &quot;Usar Microsoft Excel&quot; si desea que los datos exportados a Excel, o &quot;Imprimir Hoja de uso del generador&quot; si se utiliza el complemento opcional en el programa generador de informes que permitan la generación de informes personalizados de hecho. </li><li> Haga clic en las casillas de los elementos que desea incluir en el informe. </li><li> Para guardar todos los parámetros seleccionados en las diferentes pestañas de la &quot;Método de Análisis&quot;, haga clic de nuevo en la pestaña &quot;General&quot; y guardarlas en un nombre de archivo y en un lugar donde se puede encontrar para su uso futuro, y haga clic en el &quot; Aceptar &quot;para cerrar esta ventana. </li><li> Esto completa el ajuste de los parámetros de prueba del instrumento. </li><li> Ahora vamos a configurar el lector de microplacas. </li><li> Haga clic en el <img src="/files/ftp_upload/2465/2465icon2.jpg" alt="Icon 2" /> botón, lo que traerá la ventana de Configuración de TPM en la parte superior. </li></ul> Seleccione los pozos de las normas y las muestras en la configuración del MPR ventana / pestaña Plate, como se muestra en la Figura 7. <img src="/files/ftp_upload/2465/2465fig7.jpg" alt="Figura 7" /> Figura 7. MPR ventana de Configuración, la ficha de la Plata. <ul><li> Haga clic en la posición A1 y arrastre el ratón para E1, a continuación, haga clic en el <img src="/files/ftp_upload/2465/2465icon3.jpg" alt="Icon 3" /> botón. Se seleccionará las posiciones que se utilizarán para las normas. Esas posiciones se resaltan en color verde. </li><li> Ahora tenemos que seleccionar F1 posiciones de H3 para las muestras. Haga clic y arrastre el ratón a partir de la posición de F1, a la posición de H3 y luego haga clic en el <img src="/files/ftp_upload/2465/2465icon4.jpg" alt="Icon 4" /> botón, se destacan las posiciones en color amarillo. </li><li> A continuación, repita la misma operación para las posiciones de A2 para el E3, para seleccionar las posiciones para las mediciones de la muestra. </li><li> Ahora, vamos a cargar el archivo separado por comas variable (csv) preparado de antemano con las normas y la información de la muestra. </li></ul> Haga clic en la &quot;información bien&quot; ficha de la &quot;Ventana de Configuración de TPM&quot;, como se muestra en la Figura 8. <img src="/files/ftp_upload/2465/2465fig8.jpg" alt="Figura 8" /> Figura 8. MPR ventana de Configuración, pestaña Información del Pozo. <ul><li> A continuación, haga clic en el <img src="/files/ftp_upload/2465/2465icon5.jpg" alt="Icono de 5" /> botón. Una ventana del explorador de Windows se abrirá el cual, como se muestra en la Figura 9, que permitirá localizar el &quot;Bien information.csv&quot; archivo y lo carga. <img src="/files/ftp_upload/2465/2465fig9.jpg" alt="Figura 9" /> Figura 9. Cargando información de la muestra así. Después de cargar el &quot;Bien de información&quot; del archivo, la &quot;información bien&quot; se verá como se muestra en la Figura 10 <img src="/files/ftp_upload/2465/2465fig10.jpg" alt="Figura 10" /> Figura 10. Cargado de información muestra. </li></ul></li></ol></li></ol> 2. Ensayo Picogreen Preparación de la muestra <ol><li> Preparar 1X buffer TE (10 mM Tris-HCl, 1 mM EDTA) mediante la adición de 1 ml de tampón 20 X TEa 19 ml dl H 2 O. </li><li> Prepare la solución de Reactivo PicoGreen añadiendo 50 L 200 X reactivo PicoGreen a 9,95 ml de tampón 1 x TE. </li><li> Elaborar normas (Lambda dsDNA) al hacer diluciones seriadas de ADN de doble cadena de valores (100 mg / mL). En primer lugar se prepara una solución de 50 mg / ml dsDNA por la adición de 25 l de ADN de doble cadena de valores (100 mg / mL) y 25 l 1 x tampón TE. A continuación, preparar las soluciones estándar mediante la adición de los volúmenes indicados de ADN de doble cadena y el tampón que se enumeran en la siguiente tabla: <table border="1"><tbody><tr><td> Concentración de ADN de doble cadena </td><td> Volumen de ADN de doble cadena </td><td> Volumen de Buffer </td></tr><tr><td> 1 mg / ml </td><td> L 20 de 50 ug / ml dsDNA </td><td> 980 l 1 x tampón TE </td></tr><tr><td> 0,1 mg / ml </td><td> 100 L de 1 mg / ml de ADN de doble cadena </td><td> 900 l 1 x tampón TE </td></tr><tr><td> 0,01 mg / ml </td><td> 100 L de 0,1 mg / ml de ADN de doble cadena </td><td> 900 l 1 x tampón TE </td></tr><tr><td> 0,001 mg / ml </td><td> 100 L de 0,01 mg / ml dsDNA </td><td> 900 l 1 x tampón TE </td></tr></tbody></table></li></ol> 3. Medición de Estándares y de fluorescencia de muestras <ol><li> Pipetear 150 L de cada patrón en los pozos A1-E1 de una placa y 96, por la figura 7, y de acuerdo con la siguiente tabla: <table border="1"><tbody><tr><td> Bien </td><td> Estándar </td></tr><tr><td> A1 </td><td> 1 mg / ml </td></tr><tr><td> B1 </td><td> 0,1 mg / ml </td></tr><tr><td> C1 </td><td> 0,01 mg / ml </td></tr><tr><td> D1 </td><td> 0,001 mg / ml </td></tr><tr><td> E1 </td><td> 1 x tampón TE </td></tr></tbody></table></li><li> Pipeta de 150 muestras l desconocido en los pozos de F1 a H3, que se muestra en la Figura 7 </li><li> Vierta la solución PicoGreen preparado en el paso 1.2 en un canal diseñado para su uso pipeta multicanal. Utilice una pipeta multicanal para entregar 150 l solución PicoGreen a los pozos A1-H3. Mezcle la solución y las normas suavemente con una pipeta. </li><li> Coloque la placa en un lugar oscuro y esperar 2-5 minutos para la reacción a desarrollar. </li></ol> 4. Resultados representante Una buena curva de calibración se evalúa con los diferentes parámetros proporcionados por el software F-7000 en la medición de las normas y el cálculo de la curva de calibración por software. El coeficiente de determinación indica la bondad del ajuste de los estándares de medida y la curva de calibración calculado. Cuanto más cerca de este valor es &quot;1&quot;, el mejor es el ajuste de los valores medidos y la curva de calibración. Si el valor está lejos de ser &quot;1&quot;, las normas deben medirse de nuevo, preparado de nuevo, o cambiar la aptitud de la curva de calibración. La figura 11 muestra un ejemplo de una curva de calibración con un coeficiente de determinación calculado = 0,99993, que se obtiene para la cuantificación de ADN de doble cadena utilizando PicoGreen tinte. <img src="/files/ftp_upload/2465/2465fig11.jpg" alt="Figura 11" /> Figura 11. Ejemplo de curva de calibración de ADN de doble cadena.

<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 y Kendra L. Cox son empleados de Hitachi Tecnologías de la América del Alto que produce el instrumento utilizado en este artículo.

Pipeteo cuidado durante la preparación de muestras, así como el uso de un espectrofotómetro de fluorescencia estable y sensible es esencial para obtener buenos resultados y reproducible. En el caso del espectrofotómetro de fluorescencia utilizado para esta prueba, se sugiere con 300 l de volumen final de la muestra en el lector de microplacas. Un menor volumen disminuye la sensibilidad, y un mayor volumen puede causar la contaminación cruzada entre los pozos.

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<td>Distilled H2O</td>
<td>Reagent</td>
<td>&nbsp;</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>
<td>Supply</td>
<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>
<td>&nbsp;</td>
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<td>10 mL serological pipet</td>
<td>Supply</td>
<td>&nbsp;</td>
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quantification of dsdna using the hitachi f-7000 fluorescence spectrophotometer and picogreen dye

La cuantificación de ADN, especialmente en pequeñas concentraciones, es una tarea importante con una amplia gama de aplicaciones biológicas incluyendo ensayos estándar de biología molecular como la síntesis y purificación del ADN, las aplicaciones de diagnóstico, tales como la cuantificación de los productos de amplificación de ADN, y la detección de moléculas de ADN en las drogas preparativos. En este video vamos a demostrar la capacidad del espectrofotómetro Hitachi F-7000 de fluorescencia equipado con un accesorio lector de placas Micro para llevar a cabo la cuantificación dsDNA utilizando sondas moleculares Quant-que PicoGreen kit de reactivos de tinte. El espectrofotómetro de fluorescencia F-7000 ofrece una alta sensibilidad y mediciones de alta velocidad. Es un sistema altamente flexible, capaz de medir la fluorescencia, luminiscencia y fosforescencia. Varios modos de medición están disponibles, incluyendo la longitud de onda de escaneo, el tiempo de exploración, la fotogrametría y la 3-D de medición de análisis. El espectrofotómetro tiene una sensibilidad en el rango de 50 picomoles de fluoresceína cuando se utiliza un volumen de muestra de 300 l en el micro, y es capaz de medir velocidades de escaneo de 60.000 nm / minuto. También cuenta con un amplio rango dinámico de hasta 5 órdenes de magnitud que permite el uso de curvas de calibración en un amplio rango de concentraciones. El sistema óptico utiliza toda la óptica reflectante para la máxima energía y sensibilidad. El rango de longitud de onda estándar es de 200 a 750 nm, y se puede ampliar a 900 nm cuando se utiliza uno de los fotomultiplicadores opcional infrarrojo cercano. El sistema permite el control de temperatura opcional para el lector de placas de 5 a 60 grados Celsius, usando un control de temperatura opcional externa recirculación de líquidos. El lector de microplacas permite el uso de 96 microplacas bien, y la medición de la velocidad de 96 pozos es menos de 60 segundos cuando se utiliza el modo de cinética. Controles de software para el F-7000 y lector de microplacas son también muy flexible. Las muestras se pueden configurar en cualquiera de los formatos de columna o fila, y cualquier combinación de los pozos pueden ser elegidos para las mediciones de la muestra. Esto permite una utilización óptima de la microplaca. Además, el software permite importar configuraciones de muestra micro placa creada en Excel y se guarda en valores separados por comas, o en formato &quot;csv&quot;. Configuraciones de microplacas de medición se pueden guardar y recordar por el software para mayor comodidad y mayor productividad. Resultados de los datos se pueden enviar a un informe estándar, de Excel, o un programa Report Generator opcional.

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

Demostración de la cuantificación de ADN de doble cadena utilizando sondas moleculares PicoGreen tinte y Hitachi F-7000 Espectrofotómetro de fluorescencia equipado con un accesorio lector de microplacas.

La cuantificación de ADN de doble cadena con el Hitachi F-7000 espectrofotómetro de fluorescencia y Dye PicoGreen

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

Research

JoVE Journal

Biology

24.4K vistas.  Hitachi High Technologies America. Demostración de la cuantificación de ADN de doble cadena utilizando sondas moleculares PicoGreen tinte y Hitachi F-7000 Espectrofotómetro de fluorescencia equipado con un accesorio lector de microplacas.

Video: 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.

Concentración de proteínas microvolumen Determinación mediante el NanoDrop 2000c Espectrofotómetro

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

Investigación

Biología

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.

Time-lapse de imágenes de mitosis después de la transfección de 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.

La cuantificación de γH2AX focos en respuesta a la radiación 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.

Determinación de la concentración de ácidos nucleicos y proteínas utilizando el micro-volumen Bio-spec Espectrofotómetro 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.

Absoluta Quantum medición del rendimiento de las muestras de polvo

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.

La visualización de Caenorhabditis Elegans Estructuras de la cutícula uso del colorante lipofílico 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.

Visualización y Análisis de las moléculas de ARNm mediante fluorescencia<em&gt; In Situ</em&gt; La hibridación en<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).

La observación y cuantificación de los telómeros y las secuencias repetitivas usando fluorescencia<em&gt; In Situ</em&gt; La hibridación (FISH) con sondas de PNA en<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.

Visualización y cuantificación de tejidos adiposos marrones y beige en ratones utilizando [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.

Cuantificación de la competencia interbacteriana utilizando imágenes de fluorescencia unicelular

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