ES2648798T3 - Materiales de captura de polinucleótidos y métodos de utilización de los mismos - Google Patents

Materiales de captura de polinucleótidos y métodos de utilización de los mismos Download PDF

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ES2648798T3
ES2648798T3 ES08826342.1T ES08826342T ES2648798T3 ES 2648798 T3 ES2648798 T3 ES 2648798T3 ES 08826342 T ES08826342 T ES 08826342T ES 2648798 T3 ES2648798 T3 ES 2648798T3
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Sundaresh N. Brahmasandra
Elizabeth Boutt
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Abstract

Un método para aislar polinucleótidos desde una muestra que contiene células, comprendiendo dicho método: contacto de la muestra con una solución de lisis y una pluralidad de partículas de unión revestidas en moléculas de PAMAM (Generación 0) covalentemente unidas con grupos carboxílicos en la superficie de las partículas de unión, donde cada moléculas de PAMAM (Generación 0) incluye exactamente cinco grupos amina disponibles para protonación, de manera que los polinucleótidos se liberan desde las células y se unen a los polinucleótidos desde dichas células con las partículas revestidas con PAMAM (Generación 0), en virtud de lo cual se crean partículas de unión unidas con polinucleótidos y una solución que contiene materia celular residual; compactación de las partículas de unión unidas con polinucleótidos; retirada de la solución que contiene la materia celular residual; lavado de las partículas de unión y liberación de los polinucleótidos desde las partículas de unión.

Description

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puntas de pipeta 103, 123 y 133 y por lo tanto representa una reutilización de una de dichas puntas. Aunque es deseable el uso de perlas magnéticas, también se utilizan perlas no magnéticas en el presente documento y se separan, p.ej., por centrifugación en lugar de mediante el uso de un imán.
5 En ciertas realizaciones, la relación entre el volumen de la muestra original introducida en el tubo de procesamiento y el volumen de líquido en el que se libera el ARN o ADN es al menos aproximadamente 10 (p.ej., al menos aproximadamente 50, al menos aproximadamente 100, al menos aproximadamente 250, al menos aproximadamente 500, al menos aproximadamente 1,000). En algunas realizaciones, se puede retener ARN o ADN de una muestra que tiene un volumen de aproximadamente 2 ml dentro del tubo de procesamiento y liberarse tras la unión y el lavado en aproximadamente 4 microlitros o menos (p.ej., aproximadamente 3 microlitros o menos, aproximadamente 2 microlitros o menos, aproximadamente 1 microlitros o menos) del líquido.
En algunas realizaciones, la muestra tiene un volumen mayor que el volumen concentrado de las partículas de unión que tienen unido en ellas ARN o ADN en un factor de al menos aproximadamente 10.
15 En otras realizaciones, la muestra tiene un volumen de 100 μl – 1 ml y las partículas compactadas ocupan un volumen efectivo de menos de 2 microlitros.
El líquido en el que se libera ARN o ADN incluye normalmente al menos aproximadamente 50 % (p.ej., al menos aproximadamente 75 %, al menos aproximadamente 85 %, al menos aproximadamente 90 % o al menos aproximadamente 95 %) del ARN o ADN, respectivamente, presente en la muestra 109. Así por ejemplo, se pueden liberar ∼8 -10 μg de ADN desde 1 ml de un cultivo de toda la noche y se pueden extraer ∼2 -4 μg de ADN desde una torunda bucal. La concentración de ARN o ADN presente en el líquido de liberación puede ser más alta que la correspondiente concentración en la muestra original ya que el volumen del líquido de liberación es normalmente
25 menos que el volumen de la muestra líquida original. Por ejemplo, la concentración de ADN en el líquido de liberación puede ser al menos aproximadamente 10 veces mayor (p.ej., al menos aproximadamente 25 veces mayor, al menos aproximadamente 100 veces mayor) que la concentración del ADN en la muestra 109. La concentración de inhibidores presentes en el líquido en el que se libera ARN o ADN es generalmente menos que la concentración de los inhibidores de la muestra fluídica original en una cantidad suficiente como para aumentar la eficiencia de amplificación del ARN o ADN con respecto a la que se podría obtener de una muestra sin purificar.
En general, aunque los procesos y materiales descritos en el presente documento tienen capacidad para actuar perfectamente – normalmente solamente con una adaptación de rutina – en una amplia gama de tamaños de muestra y volúmenes de reactivos, para la mayoría de las aplicaciones prácticas (considerando el tamaño de la 35 mayoría de las muestras biológicas sometidas a análisis de diagnóstico), el volumen de las partículas compactadas que tienen ARN y/o ADN unidos a ellas que resulta (antes de liberación) está en el intervalo de 2 – 3 μl y es independiente del volumen de la muestra, hasta aproximadamente 2 ml de la muestra. Normalmente, la cantidad de las micropartículas requeridas se determina por la cantidad de ARN y/o ADN en la muestra. Se ha observado que, dada la eficiencia de la unión a las partículas, es suficiente 0,5 mg de partículas para la mayoría de las aplicaciones manuales y para la mayoría que implican un pipeteado automático, independientemente del tamaño de la muestra. Por lo tanto, por ejemplo, para muestras que tienen volúmenes de 0,5 microlitros a 3 microlitros, el volumen de las partículas compactadas es de 2-3 μl. Por ejemplo, para Chlamydia, el tamaño de la muestra es normalmente 1 ml, y es suficiente 0,5 mg de partículas. Para otras aplicaciones, se puede extraer también ADN de una muestra de 2 ml con 0,5 mg de partículas o, en algunos casos, se pueden usar perlas de 1 mg. Para muestras más pequeñas, como
45 por ejemplo las que tienen un volumen de 50 ml, suele ser todavía lo normal utilizar partículas de 0,5 mg.
Para agitar la solución en varias etapas durante el proceso manual, se puede pipetear la solución arriba y abajo varias veces, por ejemplo 10 veces, 15 veces o 20 veces. Dicho procedimiento es aceptable durante la etapa de liberación, así como las etapas de lavado. La agitación con vórtice también funciona para estas etapas. Sin embargo, para los procesos automáticos, es imposible tolerar ninguna etapa de mezclado, el número de operaciones de mezclado se mantiene en el mínimo ya que posiblemente fuera la causa de que parte del PAMAM(0) se saliera e inhibiera la PCR en dirección 3’.
El proceso descrito en el presente documento representa una limpieza enormemente eficaz de una muestra en la
55 preparación de PCR y proporciona la capacidad de detectar tan solo 25 copias de ARN o ADN desde 1 mililitro de muestra clínica. EL ARN o ADN está presente en un alto nivel de concentración ya que el volumen de elución puede ser de tan solo 3 microlitros. Asimismo hay un reducido líquido de muestra residual y/o volumen de lavado en las microesferas concentradas, minimizando la dilución de la muestra o el tampón de lavado, además de minimizar la inhibición de la muestra residual.
El intervalo de tiempo entre la introducción de la muestra que contiene el polinucleótido en tubo de procesamiento 101 y la liberación del ARN o ADN en el líquido de liberación está comprendido normalmente entre 10 y 30 minutos, y es normalmente aproximadamente 15 -20 minutos o puede ser 15 minutos o menos (p.ej., aproximadamente 10 minutos o menos, aproximadamente 5 minutos o menos). Estos períodos de tiempo incluyen el período de lisis (que
65 duplica el período de unión a muestra) y son enormemente rápidos. Para liberar ARN y ADN, por separado, de una sola muestra, tan solo es necesario añadir un procedimiento de liberación adicional como en 140 en la FIG. 1.
8
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EL pKa de un grupo amino se define por su base conjugada, del siguiente modo : una amina protonada, R-NH3+ está en equilibrio disociativo:
R-NH3+ ↔ H+ + R-NH2 5 y su pKa se da por –log10 Ka, donde Ka = [H+] [R-NH2] / [R-NH3+].
Dado que un átomo de nitrógeno es trivalente y debido a las condiciones de dendrimerización, cada molécula de PAMAM(0) tiene una mezcla de grupos amina primaria, terciaria. Por lo tanto, las moléculas PAMAM (0) presentan múltiples valores pKa a lo largo de un intervalo de valores aproximadamente en consonancia con el intervalo de pKa cubierto por las aminas alifáticas primarias y terciarias, cuyos pKa se encuentran en el intervalo de 10-11 normalmente, tal como lo prueba por ejemplo, en la Tabla 12.2 de Organic Chemistry, 2ª Ed., Allinger, et al., Eds., Worth Publishers, Inc. (1976). Sin embargo, de acuerdo con la información facilitada por el fabricante de PAMAM (0), Dendritech of Midland, Michigan, es probable que de hecho PAMAM tengan pKa en el intervalo de 5,5 (para las
15 aminas terciarias en el interior de la molécula) – 9,5 (para las aminas primarias en la superficie de las moléculas de PAMAM). El artículo Tomalia, et al., Angew. Chem. Int. Ed. Engl., 29, 138-175 (1990), en la página 163, columna derecha de la publicación, se hace referencia estos datos.
PAMAM(0) es eficaz como agente de unión para ADN en los procesos descritos en el presente documento al menos en parte porque los grupos amina de PAMAM(0) tienen un pKa comprendido entre 5 -9. Por tanto, a un pH bajo, normalmente está cargado positivamente – y puede llevar incluso cargas positivas múltiples por molécula que tienen lugar a partir de las protonaciones de los grupos amina a pH más bajos que su pKa – y por tanto es capaz de unirse fuertemente a los polinucleótidos, como ADN y ARN, que normalmente comprenden polianiones (de carga negativa predominantemente) en solución.
25 Durante el uso de la molécula PAMAM (0) en los procesos descritos en el presente documento, el pH del tampón de unión (normalmente TRIS) utilizado para lisar células en el mismo momento en el que se une el ADN liberado con las partículas, es aproximadamente 7-8. A este pH, todas las aminas (6 grupos posibles por cada molécula PEI, distribuidas por Sigma) permanecen protonadas (con carga positiva) y, por tanto, atraen intensamente las moléculas de ADN cargadas negativamente para su unión hacia las perlas.
Las moléculas PAMAM (0) también son ventajosas porque son resistentes, p.ej. son inmunes, a la degradación de enzimas líticas, enzimas de proteasa (p.ej. mezclas de endo-y exo-proteasas, como pronasa que escinden los enlaces péptido), sustancias químicas duras, como detergentes, y calor hasta 95 ºC, y como tales son capaces de
35 unirse a ARN y ADN durante el proceso de lisis también. Por lo tanto, la lisis de células y la unión de ARN y/o ADN se pueden combinar en una sola etapa (sincrónica), en virtud de lo cual se ahorra tiempo y al menos una etapa de procesamiento. La unión fuerte de moléculas de ARN y/o ADN a PAMAM (0) permite un rápido lavado de las perlas de afinidad revestidas en PAMAM (0) para retirar los inhibidores de PCR utilizando una solución de lavado. La retirada de ARN y/o ADN desde las perlas de afinidad se lleva a efecto elevando la temperatura en presencia de un reactivo de retirada patentado. Dado que la cantidad de las perlas utilizada es muy reducida, (< 1 μl), el ARN y/o el ADN se pueden liberar en un volumen final de tan solo 3 microlitros. El ARN y/o ADN liberado se neutraliza a un volumen final de 5 a 50 microlitros utilizando un reactivo de neutralización y queda listo entonces para PCR en dirección 3’.
45 Normalmente, la cantidad de la muestra introducida es aproximadamente 500 microlitros o menos (p.ej., aproximadamente 250 microlitros o menos, aproximadamente 100 microlitros o menos, aproximadamente 50 microlitros o menos, aproximadamente 25 microlitros o menos, aproximadamente 10 microlitros o menos). En algunas realizaciones, la cantidad de la muestra es aproximadamente 2 microlitros o menos (p.ej., aproximadamente
0.5 microlitros o menos).
PAMAM(0) proporciona una recuperación de ARN y ADN excelente, sobre la base en parte de su alta capacidad de unión y su alta eficiencia de liberación. En general, la relación entre la masa de partículas y la masa de ARN o ADN retenida por las partículas no es más de aproximadamente 25 o más (p.ej., no más de aproximadamente 20, no más de aproximadamente 10). Por ejemplo, En algunas realizaciones, aproximadamente 1 gramo de partículas retiene
55 aproximadamente 100 miligramos de ARN o ADN; cuando se utiliza en cantidades más reducidas, se pueden obtener relaciones similares (p.ej., una capacidad de unión de ∼100 mg de ARN o ADN /mg perlas).
Otro aparato para captura de ADN
En otros ejemplos, se puede configurar el soporta sólido como un elemento de retención (p.ej., un elemento poroso como pueda ser una columna, un filtro, una membrana porosa, un filtro microporoso o una matriz de ge que tiene múltiples aperturas, como poros y/o canales, a través de los cuales pasa el ARN y/o ADN), a través del cual debe pasar el material de muestra (que contiene ARN y/o ADN). Dicho elemento de retención puede estar formado de múltiples partículas modificadas en su superficie restringidas en una geometría adecuada. En algunos ejemplos, el 65 elemento de retención comprende una o más membranas de filtro disponibles, por ejemplo, de Osmonics, que se forman de polímeros que pueden estar modificados en su superficie también y utilizarse para retener ARN y/o ADN.
13
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tampón y plasma. Se utilizaron 500 copias de ARN de EV13 por 1 ml tanto en las muestras de tampón como las de plasma de acuerdo con un proceso, tal como se describe con mayor detalle en el presente documento.
La FIG. 6 muestra la extracción de ADN, utilizando perlas de PAMAM (0). Se extrajeron 2,5 pg ADN con adiciones 5 en un tampón M4, o un tampón de lisis tal como se describe en el presente documento utilizando el proceso para extraer ARN de un tampón de recogida M4 tal como se describe con mayor detalle en el presente documento..
La FIG. 7 muestra las curvas de PCR para la extracción de ARN desde una muestra de plasma de 500 μl que contiene 200 copias de ARN EV13, utilizando tratamiento con 7U ADNasa.
10 LA FIG. 8 muestra ejemplos de la sensibilidad del proceso. El análisis Probit revela una LOD de 50 copias /200 ml LCR.
Ejemplo 4: Ejemplo de protocolo para la extracción de ARN desde M4, torunda seca en 1X tampón TCEP, 15 muestras THB
Proceso previo de preparación de muestra (solamente requieren filtración las muestras de torunda)
Etapa
Acción
1
Pipetear 500 μl de la muestra en un tubo (tubo con tapón a presión 1,7 ml DOT) que contiene 500 μl de tampón TCEP
2
Pipetear de arriba a abajo 2x, y a continuación pipetear la cantidad entera en la jeringuilla de 3 ml
3
Insertar el émbolo en la jeringuilla y filtrar el contenido en un tubo limpio (tubo con tapón a presión 1,7 ml DOT), aplicando presión hasta que se expulsa el líquido y empieza a salir la espuma del filtro (evitar que la espuma llegue a la muestra).
Extracción de ARN y preparación PCR
Etapa
Acción
1
Pipetear muestra (500 μl de muestra, más 500 μl de tampón TCEP) en un tubo con tapón a presión 1,7 ml DOT que contiene 30 μl de perlas magnéticas de ARN. Cerrar e invertir el tubo de reacción 5 veces o hasta que se dispersan las perlas (o se disuelven en el caso de perlas liofilizadas).
2
Inmediatamente colocar las muestras en un baño de agua a 60 °C, e incubar durante 10 min (10,5 minutos, máximo).
3
Retirar muestras del baño de agua y secar la parte exterior del tubo con un paño absorbente.
4
Colocar los tubos sobre una rejilla magnética y permitir que tenga lugar la separación durante 1 minuto (1,5 minutos max).
5
Utilizando una pipeta nueva para cada muestra, aspirar cuidadosamente 1 ml de sobrenadante de cada muestra (sin perturbar las perlas), utilizando una pipeta de 1 ml. Descartar el sobrenadante. Asegurarse de retirar cualquier líquido que quede en el tapón del tubo.
6
Tras la retirada del sobrenadantes inicial de todas las muestras, retirar cualquier líquido que quede utilizando una punta pipeta de 1 ml nueva para cada muestra.
7
Colocar tubos en una rejilla para tubos no magnética y añadir 100 μl de tampón de lavado (0,1 mM Tris, pH 7,5) a cada tubo utilizando una punta de pipeta de 200 ul. Pipetear de arriba a abajo 10 veces, o hasta resuspender todas las perlas magnéticas y que no quede pegada ninguna perla en la punta de la pipeta.
8
Colocar los tubos sobre la rejilla magnética durante 30 segundos, dejando que se separen las perlas.
9
Aspirar cuidadosamente el sobrenadante de todas las muestras utilizando una punta de pipeta de 200 ul. Descartar el sobrenadante. Utilizando una punta nueva de 20 ul para cada muestra, aspirar cualquier líquido que quede en la muestra (es decir, líquido que se ha "sedimentado" tras la primera etapa de aspiración) y descartar el líquido.
10
Colocar los tubos en una rejilla de tubos no magnética y añadir 10 μl de Tampón de liberación (20 mM Bis-Tris Propano o 20 mM Tris pH 9). Agitar con vórtice durante 10 segundos o hasta resuspender las perlas.
Etapa
Acción
11
Colocar las muestras en un bloque de calor a 85 °C durante 3 minutos (3,5 minutos max.).
12
Retirar las muestras del bloque de calor y colocar encima de la rejilla magnética durante 30 segundos (1 minutos max.).
13
Manteniendo los tubos sobre la rejilla magnética, retirar todo el líquido, evitando cuidadosamente las perlas magnéticas a un lado del tubo y colocar en un tubo 0,65 ml DOT.
14
Mezclar la muestra pipeteando de arriba a abajo una vez. La muestra queda lista ahora para PCR.
15
Preparar la mezcla de PCR utilizando un kit Quantitect con adiciones de cebadores 0,6 uM y extra platinum taq.
16
Añadir 8 ul de la mezcla en instrumentos capilares Rotorgene o LC, añadir 2 ul de ARN.
16
Etapa
Acción
17
Poner en marcha el programa RT PCR del siguiente modo: 50 grados durante 20 min (etapa a temperatura ambiente), 95 grados durante 5 min (desnaturalización), ciclar a 95-2 s, 58-50 s (50 ciclos)
Ejemplo 5: Ejemplo de protocolo para la extracción de ARN desde muestras de plasma
Extracción de ARN y preparación PCR
Etapa
Acción
1
Pipetear la muestra (500 μl de muestra, más 500 μl de tampón TCEP, 70 μl 10 % SDS) en un tubo con tapón a presión 1.7 ml DOT que contiene 30 ml de perlas magnéticas de ARN. Cerrar e invertir el tubo de reacción 5 veces o hasta que se dispersan las perlas (o se disuelven en el caso de perlas liofilizadas).
2
Inmediatamente colocar las muestras en un baño de agua a 60 °C, e incubar durante 10 min (10,5 minutos, máximo).
3
Retirar las muestras del baño de agua y secar la parte exterior del tubo con un paño absorbente.
4
Colocar los tubos sobre una rejilla magnética y permitir que tenga lugar la separación durante 1 minuto (1,5 minutos max).
5
Utilizando una pipeta nueva para cada muestra, aspirar cuidadosamente 1 ml de sobrenadante de cada muestra (sin perturbar las perlas), utilizando una pipeta 1 ml. Descartar el sobrenadante. Asegurarse de retirar cualquier líquido que quede en el tapón del tubo.
6
Tras la retirada del sobrenadantes inicial de todas las muestras, retirar cualquier líquido que quede utilizando una punta pipeta de 1 ml nueva para cada muestra.
7
Añadir 250 ul de tampón ADNasa con 1 aglomerado de ADNasa o 5 unidades de ADNasa líquida. Resuspender las perlas por agitación con vórtice o pipeteado.
8
Se incuba a 37 durante 10 min.
9
Colocar los tubos sobre una rejilla magnética y permitir la separación para proceder durante 1 minuto (1,5 minuto max).
10
Utilizando una pipeta nueva para cada muestra, aspirar cuidadosamente 1 ml de sobrenadante de cada muestra (sin perturbar las perlas), utilizando una pipeta de 1 ml. Descartar el sobrenadante. Asegurarse de retirar cualquier líquido que quede en el tapón del tubo.
11
Colocar tubos en una rejilla para tubos no magnética y añadir 100 μl de tampón de lavado (0,1 mM Tris, pH 7,5) a cada tubo utilizando una punta de pipeta de 200 ul. Pipetear de arriba a abajo 10 veces, o hasta resuspender que todas las perlas magnéticas y que no queda pegada ninguna perla en la punta de la pipeta.
12
Colocar los tubos sobre la rejilla magnética durante 30 segundos, dejando que se separen las perlas.
13
Aspirar cuidadosamente el sobrenadante de todas las muestras utilizando una punta de pipeta de 200 ul. Descartar el sobrenadante. Utilizando una punta nueva de 20 ul para cada muestra, aspirar cualquier líquido que quede en la muestra (es decir, líquido que se ha "sedimentado" tras la primera etapa de aspiración) y descartar el líquido.
14
Colocar los tubos en una rejilla de tubos no magnética y añadir 10 μl de Tampón de liberación (20 mM Bis-Tris Propano o 20 mM Tris pH 9). Agitar con vórtice durante 10 segundos o hasta resuspender las perlas.
15
Colocar las muestras en un bloque de calor a 85 °C durante 3 minutos (3,5 minutos max.).
16
Retirar las muestras del bloque de calor y colocar encima de la rejilla magnética durante 30 segundos (1 minutos max.).
17
Manteniendo los tubos sobre la rejilla magnética, retirar todo el líquido, evitando cuidadosamente las perlas magnéticas a un lado del tubo y colocar en un tubo 0,65 ml DOT.
18
Mezclar la muestra pipeteando de arriba a abajo una vez. La muestra queda lista ahora para PCR.
19
Preparar la mezcla de PCR utilizando un kit Quantitect con la adición de cebadores 0,6 uM y extra platinum taq.
20
Añadir 8 ul de la mezcla en instrumentos capilares Rotorgene o LC, añadir 2 ul de ARN.
21
Poner en marcha el programa RT PCR del siguiente modo: 50 grados durante 20 min (etapa a temperatura ambiente), 95 grados durante 5 min (desnaturalización), ciclar a 95-2 s, 58-50 s (50 ciclos)
5
Ejemplo 6: Proceso de ensamblaje para 2X Tampón TCEP para extracciones de ARN
El procedimiento en este ejemplo proporciona un método apropiado para preparar 50 ml de 2 x tampón TCEP (20 mM Tris HCl pH 7,0, 2 % Tx-100, 10 mM TCEP) utilizado en extracciones de ARN, tal como se describe en el 10 presente documento con mayor detalle. A continuación, se ofrece una lista de los reactivos utilizados en el proceso.
• 1 M Tris-HCl pH 7,0
17
imagen13
imagen14
Etapa
Acción
3
Etiquetar tubos cónicos 1 -50 ml con número de lote, fecha e iniciales
4
Pipetear la cantidad apropiada de microesferas en un tubo cónico de 50 ml
5
Colocar los tubos cónicos en una rejilla magnética y dejar asentar hasta que las perlas son capturadas completamente por el imán. Retirar sobrenadante cuidadosamente
Lavado tampón MES
Durante 30 minutos
6
Añadir Tampón SN-B a cada tubo y agitar con vórtice para mezclar Colocar los tubos cónicos en una rejilla magnética y dejar reposar hasta que se capturan las perlas completamente con el imán. Retirar el sobrenadante cuidadosamente. repetir el lavado 2 veces más 10 minutos
Preparar sulfo-NHS
7
Pesar una pequeña cantidad de sulfo-NHS balanza para papel y multiplicar el peso (en mg) por 20 para calcular los μl de agua ultra pura que se deben añadir para obtener una solución de 50 mg/ml. Se necesitan 1,5 ml para 1 reacción (75 mg). Añadir a un tubo de 1,7 ml y mezclar bien. Pesar (mg) X 20 = μl agua ultra pura necesaria. Añadir agua ultra pura y agitar con vórtice hasta que se resuspende. Se deberá preparar la solución NHS inmediatamente antes de su uso y descartar al cabo de 15 min
Activación (Preparar EDAC en campana)
8
Añadir los reactivos en el siguiente orden a cada tubo cónico Durante 30 minutos
(i) ddH2O
5000 ml
(ii) Tampón SN-C
1000 ml
(iii) 50 mg/ml sulfo-NHS
1500 ml
Sonicar utilizando un desmembrador ultrasonido a una potencia de 12 durante un período apropiado asegurándose de que la sonda está sumergida en todas las ocasiones. Limpiar la sonda con H2Od y secar con un trapo antes de la sonicación
10 segundos
9
Inmediatamente preparar 5 mg/ml EDAC en la campana. Se deberá preparar la solución de EDAC inmediatamente antes de su uso, descartar al cabo de 15 min. Pesar una pequeña cantidad de EDAC en papel de peso y multiplicar el peso (en mg) por 200 para calcular los μl de agua ultra pura necesarios para obtener una solución de 5 mg/ml. Se necesitan 2480 ml en total (12,4 mg). Preparar en tubos cónicos de 50 ml. Pesar (mg) X 200 = (ml) agua ultra pura que se ha de añadir Agitar con vórtice a fondo después de añadir H2O ultra pura
10
Añadir los siguientes: (i) 10% Triton X-100 (ii) 5 mg/ml EDAC (añadir solución de EDAC cuidadosamente; gota a gota mientras se agita con vórtice la solución a una velocidad muy baja). 10 ml 2480 ml
Activación (Preparar EDAC en campana)
(iii) 50 mg/ml sulfo-NHS
1500 ml
Mezclar bien con vórtice
11
Registrar el número de veces que EDAC abrió el frasco. Descartar tras 5 usos.
12
Asegurar los tubos para el agitador orbital con cinta de etiquetado e incubar a temperatura ambiente en el ajuste 6 (o en un ajuste en el que las microesferas se mezclan bien) 30 minutos.
13
Tras la incubación, centrifugar durante 5 min a una velocidad máxima y a continuación colocar en el imán. Retirar el sobrenadante cuidadosamente pero completamente 5 minutos
Tampón de lavado MES
Durante 30 minutos
14
Añadir tampón SN-B a cada tubo y agitar con vórtice para mezclar Colocar tubos cónicos en una rejilla magnética y dejar en reposo hasta que las perlas son capturadas totalmente por el imán. Retirar el sobrenadante cuidadosamente. Repetir el lavado 2 veces más. 10 minutos
Acoplamiento
15
Preparar reacción de acoplamiento Durante 10 minutos
(i) Añadir Tampón H
6 ml
20
imagen15

Claims (1)

  1. imagen1
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