Mitochondrial Genome DNA Extraction Kit (for Animals)

1. Componentes del kit de reactivos

2. Almacenamiento

This kit can be stored at room temperature (15-25℃) in dry conditions for 12 meses. The DNA extraction purification columns can be stored in a cool and dry environment for 1 año. Proteinasa K y ARNasa A contienen conservantes, permitiendo el transporte a temperatura ambiente, pero para almacenamiento a largo plazo, deben mantenerse a -20 ℃. Reagent buffers E1/E2 should be stored at 4℃.

3. Instrucciones para usar el kit de reactivos

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

3.2 Algunos componentes del kit de reactivos contienen irritantes.. Se recomiendan medidas de protección como el uso de ropa y gafas protectoras..

3.3 Durante el uso de este kit de reactivos, una centrífuga de alta velocidad, baño de agua (baño de metal), mezclador Vortex, etanol anhidro, PBS,agua desionizada estéril, y los tubos EP deben ser preparados por el usuario.

4. Introducción al kit de reactivos

The Mitochondrial Genome Purificación de ADN Kit offers a rapid and efficient method for purifying DNA from animal tissues and cell cultures. Widely used in both animal tissue and cell cultures, this kit consists of two parts. The first part involves rapid collection of high-purity animal tissue and cell mitochondria using gradient centrifugation. The second part utilizes a column-based method for quick extraction of mitochondrial DNA without the need for toxic reagents like phenol-chloroform. El ADN extraído se puede utilizar directamente para PCR, transferencia del sur, y otras aplicaciones.

5. Principios y procedimientos experimentales

6. Proceso de extracción

Antes de comenzar el experimento:

A. Reagent Buffers B and C tend to precipitate under low-temperature conditions. It is recommended to heat them at 65°C for 5 minutes until the precipitates dissolve before normal use.

B. Before using Tampón de lavado 1, follow the instructions on the reagent bottle label to add the specified amount of absolute ethanol. Entonces, check the label to indicate that ethanol has been added.

C. El tampón de elución es un 0.1x solución TE con contenido mínimo de EDTA. Si el EDTA afecta a experimentos posteriores, it is suggested to use sterile deionized water instead of the elution buffer.

D. Mitochondrial Collection: The first part involves collecting mitochondria, where centrifugation is crucial. During the extraction process, centrifugal force is expressed in ‘g’. Most centrifuges have a rpm/g conversion mode, so please pay attention.

1. Procesamiento de muestras:

A. Take cell culture medium, centrifuge at 1000g for 1 minuto para recolectar células. Try to remove as much supernatant as possible. Agregar 1ml PBS (PH7.9) to wash the cells, centrifuge to collect them, Luego añade 1ml pre-cooled (0℃) Reagent Buffer E1 to fully suspend the cells and homogenize rapidly 5-10 veces.

B. Cut tissues not exceeding 200 mg into small pieces, agregar 1ml PBS (PH7.9) for washing and absorb excess liquid with filter paper. Agregar 1ml pre-cooled (0℃) Reagent Buffer E1 and grind in an ice bath about 20 veces.

2. Transfer the liquid mixture to a centrifuge tube, centrifugar en 4℃, 1000g for 5 minutos, and transfer the supernatant to a new centrifuge tube.

(Nota: There may be precipitates in this transferred supernatant. For obtaining high-purity mitochondria, it is recommended to centrifuge and transfer the supernatant during step 2.)

3. Transfer the supernatant obtained in the previous step to a new centrifuge tube, centrifuge at 12000g, 4℃ para 10 minutos, and discard the supernatant.

(Nota: After this step, mitochondria will precipitate at the bottom of the tube.)

4. Recommended step: Agregar 5ml Reagent Buffer E2 to the mitochondrial pellet, resuspend it, centrifugar en 4℃, 1000g for 5 minutos, transfer the supernatant to a new centrifuge tube, then centrifuge at 12000g, 4℃ para 10 minutos, descartar el sobrenadante, and obtain high-purity mitochondrial precipitates at the tube bottom.

(Nota: Mitochondria collection at this step is complete. You may interrupt the experiment and store mitochondria at -70℃.)

Second Part: Isolation and Purification of Mitochondrial DNA

5. Agregar 280μl of Reagent Buffer B, 10µl de RNasa A, and 10μl of Proteinase K to the centrifuge tube containing mitochondria. Thoroughly invert to mix, digest at 65℃ para 5 minutos.
6. Agregar 300μl of Reagent Buffer C al lisado y mezclar bien. Si aparece un precipitado blanco, se puede dejar tranquilo; its disappearance will not affect subsequent experiments.
7. Agregar 300μl of absolute ethanol y mezclar bien. It may cause precipitation, pero no afectará los experimentos posteriores..
8. Transfer the obtained liquid to the DNA extraction purification column (colocar) (aproximadamente 650~700μl cada vez). Centrifuge at more than 8,000 rpm para 1 minuto, desechar los residuos recogidos, y vuelva a insertar el tubo de recolección en la columna de purificación para el siguiente paso.
9. Coloque la columna de purificación de extracción de ADN. (colocar) en un tubo de recolección, agregar 300μl de tampón de lavado 1, centrifuge at more than 8,000 rpm para 1 minuto, desechar los residuos, y vuelva a insertar la columna de purificación de extracción de ADN. (colocar) en el tubo para el siguiente paso.

(Nota: Confirm that absolute ethanol has been added to Wash Buffer 1.)

10. Agregar 400μl de tampón de lavado 1 a la columna de purificación de extracción de ADN (colocar), centrifugar en 14,000 rpm (20,000×g) para 2 minutos. Extend centrifugation time appropriately for a drier membrane.
11. Coloque la columna de purificación de extracción de ADN. (colocar) in a new centrifuge tube, open the cap, and incubate at 65℃ para 2 minutos. Extend this step as needed to evaporate ethanol completely to prevent residual ethanol from affecting downstream experiments.
12. Drip-suspend 50-100μl de tampón de elución sobre la membrana, centrifugar en 12,000 rpm para 2 minutos.

(Nota: 1. Eluting DNA with 50μl of Elution Buffer can increase DNA concentration but reduce the total yield; 2. The eluted DNA wash can be reapplied to the DNA extraction purification column for an additional collection at 12,000 rpm para 2 minutes to increase DNA yield.)