Mitochondrial Genome DNA Extraction Kit (for Animals)

1. Komponente kompleta reagensa

Tehnički podaci	50T	100T
Mačka. Ne.	SN0237	SN0238
Stupovi za ekstrakciju DNK (set)	50 (set)	100 (set)
Reagent Buffer E1	50ml	2x50ml
Reagent Buffer E2	30ml	2x30ml
Pufer reagensa b	20ml	2x20ml
Pufer reagensa c	30 ml	30 ml
Pufer za pranje 1	15 ml	2 × 15 ml
Elucijski pufer	20 ml	20 ml
Proteinaza K	1ml	1ml
RNaza A	1ml	1ml
Priručnik za upute	1	1

2. Skladištenje

This kit can be stored at room temperature (15-25℃) in dry conditions for 12 mjeseca. The DNA extraction purification columns can be stored in a cool and dry environment for 1 godina. Proteinase K and RNaza A contain preservatives, omogućavajući prijevoz na sobnoj temperaturi, Ali za dugoročno skladištenje, Treba ih držati na -20 ℃. Reagent buffers E1/E2 should be stored at 4℃.

3. Upute za korištenje kompleta reagensa

3.1 Ovaj komplet reagensa namijenjen je istraživanju molekularne biologije i ne smije se koristiti za dijagnozu ili liječenje bolesti.

3.2 Neke komponente u kompletu reagensa sadrže iritante. Preporučuju se zaštitne mjere poput nošenja zaštitne odjeće i naočala.

3.3 Tijekom upotrebe ovog kompleta reagensa, brza centrifuga, vodena kupelji (metalna kupka), vrtlog miksera, bezvodni etanol, PBS,sterilna deionizirana voda, i EP epruvete treba pripremiti korisnik.

4. Uvod u komplet reagensa

The Mitochondrial Genome Pročišćavanje DNK 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, Južni blot, and other applications.

5. Eksperimentalni principi i postupci

6. Ekstrakcijski postupak

Prije početka eksperimenta:

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 Pufer za pranje 1, follow the instructions on the reagent bottle label to add the specified amount of absolute ethanol. Zatim, check the label to indicate that ethanol has been added.

C. Buffer za eluciju je a 0.1X TE otopina 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. Dodati 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 vrijeme.

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

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

(Bilješka: 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℃ za 10 minuta, and discard the supernatant.

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

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

(Bilješka: 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. Dodati 280μl reagens pufera b, 10μl of RNase A, and 10μl of Proteinase K to the centrifuge tube containing mitochondria. Thoroughly invert to mix, digest at 65℃ za 5 minuta.
6. Dodati 300μl reagens pufera c to the lysate and mix well. Ako se pojavi bijeli talog, it can be left undisturbed; its disappearance will not affect subsequent experiments.
7. Dodati 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 za 1 minuta, discard the collected waste, and re-insert the collection tube into the purification column for the next step.
9. Postavite stupac za pročišćavanje ekstrakcije DNK (set) into a collection tube, dodati 300μl pufera za pranje 1, centrifuge at more than 8,000 RPM za 1 minuta, odbaciti otpad, and re-insert the DNA extraction purification column (set) into the tube for the next step.

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

10. Dodati 400μl pufera za pranje 1 na stupac za pročišćavanje DNK (set), centrifuga na 14,000 rpm (20,000×g) za 2 minuta. Extend centrifugation time appropriately for a drier membrane.
11. Postavite stupac za pročišćavanje ekstrakcije DNK (set) in a new centrifuge tube, open the cap, and incubate at 65℃ za 2 minuta. Extend this step as needed to evaporate ethanol completely to prevent residual ethanol from affecting downstream experiments.
12. Drip-suspend 50-100μl pufera za eluciju onto the membrane, centrifuga na 12,000 RPM za 2 minuta.

(Bilješka: 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 za 2 minutes to increase DNA yield.)