- Componentes del kit de reactivos
| Especificaciones | 50t | 100t |
| Gato. No. | SN0321-D | SN0322-D |
| Columnas de extracción de ARN (colocar) | 50（套） | 100（套） |
| Columnas de limpieza de ADN (colocar) | 50（套） | 100（套） |
| Extracción de ARN Buffer I | 30ml | 2× 30 ml |
| Tampón de eliminación de inhibidores | 30ml | 2×30 ml |
| Tampón de lavado 1 | 15 ml | 2×15 ml |
| Tampón de elución | 20ml | 2×20 ml |
| lisozima | 5ml | 2x5ml |
| Manual de instrucciones | 1 | 1 |
- Almacenamiento
Este kit de reactivos debe almacenarse a temperatura ambiente. (15-25℃) en un ambiente seco y puede conservarse durante 12 meses. Lysozyme contains a preservative, allowing for transportation at room temperature; sin embargo, for long-term storage, it should be stored at -20℃
- 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.
- Introducción al kit de reactivos
This RNA purification kit provides a rapid and efficient purification solution for bacterial RNA, suitable for most bacterial tissues. The kit employs DNA removal technology to effectively eliminate genomic DNA, and the resulting RNA samples typically do not require on-column digestion, reducing the risk of RNA degradation.
The RNA Fast Purification Kit allows for the extraction of total bacterial RNA (Incluyendo ARN nuclear y ARN citoplasmático.) dentro 1 hora. The extracted RNA can be directly used for RT-PCR, transferencia Northern, etc.. The entire purification process does not involve toxic reagents such as phenol-chloroform, making the RNA purification kit well-suited for various sample types.
- Principios y procedimientos experimentales
- Proceso de extracción
Precauciones antes de comenzar el experimento.:
A. Antes de usar, add the specified amount of absolute ethanol to Tampón de lavado 1 como se indica en la etiqueta del frasco de reactivo, and mark with a check to indicate the addition of absolute ethanol.
B. El tampón de elución es un 0.1x solución TE que contiene una cantidad mínima de EDTA. If EDTA may impact subsequent experiments, se recomienda reemplazar el tampón de elución con agua desionizada estéril.
- Procesamiento de muestras:
A. Gram-positive bacteria: Centrifuge the bacterial suspension at 4℃, 12,000 rpm para 2 minutos, collect bacterial cells (1.53 ml), descartar el sobrenadante, agregar 100μl Lysozyme (10mg/ml), thoroughly mix the bacterial cells, e incubar a temperatura ambiente durante 15-30 minutos.
B. Gram-negative bacteria: Centrifuge the bacterial suspension at 4℃, 12,000 rpm para 2 minutos, collect bacterial cells (1.53 ml), descartar el sobrenadante, agregar 10μl Lysozyme (10mg/ml), thoroughly mix the bacterial cells, e incubar a temperatura ambiente durante 3-5 minutos.
2.Agregar 400μl RNA Extraction Buffer I, vortex to mix thoroughly.
3. Transfer the lysate to a DNA clearance purification column, centrifugar en 13,000 rpm para 2 minutos, collect the filtrate (RNA is present in the filtrate).
4. Accurately estimate the volume of the filtrate, agregar 0.5 times the volume of absolute ethanol, mezclar bien; if precipitation occurs, it does not affect subsequent experiments.
5. Add the obtained liquid to the RNA extraction purification column (aproximadamente 650~700μl cada vez), centrifuge at more than 8,000 rpm para 1 minuto, Deseche el líquido de desechos recolectados, and reinsert the collection tube into the RNA extraction purification column for the next step.
6. Repita el paso 5, add the remaining liquid to the RNA extraction purification column, centrifuge at more than 8,000 rpm para 1 minuto, Deseche el líquido de desechos y el tubo de recolección.
7. Place the RNA extraction purification column into a new collection tube, agregar 600μl Inhibitor Removal Buffer, centrifuge at more than 8,000 rpm para 1 minuto, Deseche el líquido de desechos, and reinsert the RNA extraction purification column into the tube for the next step.
8. Agregar 700μl Wash Buffer 1 to the RNA extraction purification column, centrifugar en 14,000 rpm (20,000×g) para 2 minutos, extend the centrifugation time appropriately to ensure the membrane is thoroughly dried.
(Nota: Confirm that absolute ethanol has been added to Wash Buffer 1. The presence of ethanol has a serious impact on subsequent experiments, so membrane drying is crucial. Después de la centrifugación, ensure there is no ethanol present before elution. Discard the waste liquid and the collection tube.
After washing with Wash Buffer 1, the membrane on the RNA extraction purification column should only have a slight color. Después de la centrifugación, carefully remove the RNA extraction purification column without touching the collection tube to ensure no ethanol interference.)
- Place the RNA extraction purification column into a new centrifuge tube, drip 100 μl Elution Buffer onto the membrane, incubar a temperatura ambiente durante 5 minutos (15°C~25°C), centrifuge at more than 8,000 rpm para 1 minuto.
(Nota: Eluting RNA with 50 μl Elution Buffer can increase RNA concentration but decrease total RNA yield.)
- Repeat the previous step.
(Nota: A new centrifuge tube can be used to collect the RNA eluted the second time, or the original collection tube can be used to continue collecting RNA.)
Reseñas
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