Polysaccharide and Polyphenol Plant Total RNA (Plus) Small Quantity Extraction Kit

1. Componentes del kit de reactivos

2. Almacenamiento

This reagent kit can be stored at room temperature (15-25℃) in a dry environment and is stable for 12 meses.

3. Instrucciones para usar el kit de reactivos

3.1 Este kit está destinado a fines de investigación de biología molecular y no debe utilizarse para el diagnóstico o tratamiento de enfermedades..

3.2 Algunos componentes del kit contienen irritantes.; es recomendable tomar las precauciones necesarias (como usar ropa y gafas protectoras).

3.3 El uso de este kit requiere equipo adicional como una centrífuga de alta velocidad., baño de agua (baño de metal), mezclador Vortex, etanol anhidro, nitrógeno líquido, cloroformo, agua desionizada estéril, y tubos EP.

4. Introducción al kit de reactivos

This RNA purification kit provides a fast and efficient method for purifying polysaccharide and polyphenol plant total RNA, suitable for most polysaccharide and polyphenol-rich plant tissues. In general, plant tissues rich in polysaccharides and polyphenols contain a high level of secondary phenolic metabolites and polysaccharides, significantly affecting RNA extraction efficiency. This kit can effectively remove polysaccharides and polyphenols, as well as efficiently eliminate DNA contamination from samples. If the experiment is sensitive to DNA, it is recommended to use primers spanning introns for downstream experiments.

The RNA Fast Purification Kit can extract total RNA from plant tissues (Incluyendo ARN nuclear y ARN citoplasmático.) dentro 1 hora. The extracted RNA can be directly used for RT-PCR, transferencia Northern, etc.. Todo el proceso de purificación no requiere reactivos tóxicos como el fenol-cloroformo., making this RNA purification kit suitable for various other sample types.

5. Principios y procedimientos experimentales

6. Proceso de extracción

Precauciones antes de comenzar el experimento.:

A. Before using Tampón de lavado 1, add the specified amount of absolute ethanol according to the label on the reagent bottle, and check the box on the label to indicate that absolute ethanol has been added.

B. The Elution Buffer is a 0.1x solución TE que contiene un mínimo de EDTA. Si el EDTA afecta a experimentos posteriores, se recomienda reemplazar el tampón de elución con agua desionizada estéril.

1. Procesamiento de muestras:

A. Recolección y almacenamiento de materiales:

If fresh materials cannot be used immediately, place them in liquid nitrogen and store them at -80°C.

B. Whenever possible, collect fresh materials, as they contain fewer polysaccharides and polyphenols.

2. Grind approximately 100 mg of fresh samples or not more than 20 mg de material seco utilizando nitrógeno líquido.

3. Agregar 600μl RNA Extraction Buffer I, ensuring there are no blocky tissues in the ground sample. Blocky tissues are difficult to lyse and can reduce RNA yield.

4. Vortex for 30 segundos.

5. Centrifugar el lisado para 5 minutos a las 12,000 rpm.

(Nota: Polysaccharide plant materials may produce a lot of sticky substances at this step, which can shear RNA in subsequent steps. Por lo tanto, remove these substances during this step.)

6. Transfer the supernatant obtained in the previous step to a DNA Clear Purification Column, centrifugar en 12,000 rpm para 30 segundos, and collect the filtrate (Nota: RNA is present in the filtrate).
7. Agregar 250μl of absolute ethanol, mezclar pipeteando. If there is a small amount of precipitation, it does not affect subsequent experiments. Add the liquid to the RNA purification column, centrifugar en 12,000 rpm para 30 segundos, and discard the flow-through.
8. Agregar 700μl Inhibitor Removal Buffer a la columna de purificación de ARN, centrifugar en 12,000 rpm para 30 segundos, and discard the flow-through.
9. Agregar 700μl Wash Buffer 1 a la columna de purificación de ARN, centrifugar en 12,000 rpm para 30 segundos, and discard the flow-through.
10. Agregar 500μl Wash Buffer 1 a la columna de purificación de ARN, centrifugar en 12,000 rpm para 3 minutos, and discard the flow-through.
11. Place the RNA purification column into a new 1.5ml centrifuge tube, air-dry the membrane at room temperature for 2 minutos.

(Nota: Confirm the addition of ethanol to Wash Buffer 1. The presence of ethanol significantly affects subsequent experiments. Por lo tanto, El secado de la membrana es crucial.. Después de la centrifugación, ensure the absence of ethanol before elution. Desechar los residuos y el tubo de recogida.. Después de usar el tampón de lavado 1, the membrane on the RNA purification column should only have a slight color. Carefully remove the RNA purification column after centrifugation, ensuring it does not touch the collection tube to avoid ethanol contamination.)

12. Goteo 50-100µl de tampón de elución sobre la membrana, centrifugar en 12,000 rpm para 1 minuto, and collect the RNA solution.

(Nota: Eluting RNA with 50μl Elution Buffer can increase RNA concentration but decrease total RNA yield.)