Mitochondrial Genome DNA Extraction Kit (for Animals)

1. Components of the reagent kit

2. Storage

This kit can be stored at room temperature (15-25℃) in dry conditions for 12 months. The DNA extraction purification columns can be stored in a cool and dry environment for 1 year. Proteinase K and RNase A contain preservatives, allowing transportation at room temperature, but for long-term storage, they should be kept at -20℃. Reagent buffers E1/E2 should be stored at 4℃.

3. Instructions for Using the Reagent Kit

3.1 This reagent kit is intended for molecular biology research and should not be used for disease diagnosis or treatment.

3.2 Some components in the reagent kit contain irritants. Protective measures such as wearing protective clothing and goggles are recommended.

3.3 During the usage of this reagent kit, a high-speed centrifuge, water bath (metal bath), vortex mixer, anhydrous ethanol, PBS,sterile deionized water, and EP tubes need to be prepared by the user.

4. Introduction to the Reagent Kit

The Mitochondrial Genome DNA Purification 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. The extracted DNA can be directly used for PCR, Southern blotting, and other applications.

5. Experimental Principles and Procedures

6. Extraction Process

Before Starting the Experiment:

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 Wash Buffer 1, follow the instructions on the reagent bottle label to add the specified amount of absolute ethanol. Then, check the label to indicate that ethanol has been added.

C. Elution Buffer is a 0.1x TE solution with minimal EDTA content. If EDTA affects subsequent experiments, 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. Sample Processing:

A. Take cell culture medium, centrifuge at 1000g for 1 minute to collect cells. Try to remove as much supernatant as possible. Add 1ml PBS (PH7.9) to wash the cells, centrifuge to collect them, then add 1ml pre-cooled (0℃) Reagent Buffer E1 to fully suspend the cells and homogenize rapidly 5-10 times.

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

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

(Note: 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℃ for 10 minutes, and discard the supernatant.

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

4. Recommended step: Add 5ml Reagent Buffer E2 to the mitochondrial pellet, resuspend it, centrifuge at 4℃, 1000g for 5 minutes, transfer the supernatant to a new centrifuge tube, then centrifuge at 12000g, 4℃ for 10 minutes, discard the supernatant, and obtain high-purity mitochondrial precipitates at the tube bottom.

(Note: 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. Add 280μl of Reagent Buffer B, 10μl of RNase A, and 10μl of Proteinase K to the centrifuge tube containing mitochondria. Thoroughly invert to mix, digest at 65℃ for 5 minutes.
6. Add 300μl of Reagent Buffer C to the lysate and mix well. If a white precipitate appears, it can be left undisturbed; its disappearance will not affect subsequent experiments.
7. Add 300μl of absolute ethanol and mix thoroughly. It may cause precipitation, but it won’t affect subsequent experiments.
8. Transfer the obtained liquid to the DNA extraction purification column (set) (approximately 650~700μl each time). Centrifuge at more than 8,000 rpm for 1 minute, discard the collected waste, and re-insert the collection tube into the purification column for the next step.
9. Place the DNA extraction purification column (set) into a collection tube, add 300μl of Wash Buffer 1, centrifuge at more than 8,000 rpm for 1 minute, discard the waste, and re-insert the DNA extraction purification column (set) into the tube for the next step.

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

10. Add 400μl of Wash Buffer 1 to the DNA extraction purification column (set), centrifuge at 14,000 rpm (20,000×g) for 2 minutes. Extend centrifugation time appropriately for a drier membrane.
11. Place the DNA extraction purification column (set) in a new centrifuge tube, open the cap, and incubate at 65℃ for 2 minutes. Extend this step as needed to evaporate ethanol completely to prevent residual ethanol from affecting downstream experiments.
12. Drip-suspend 50-100μl of Elution Buffer onto the membrane, centrifuge at 12,000 rpm for 2 minutes.

(Note: 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 for 2 minutes to increase DNA yield.)