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Mitochondrial DNA Isolation Kit

Details for Product No. ABIN412491, Supplier: Log in to see
Application
DNA Isolation (DNAIso)
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Purpose Isolates mitochondrial DNA, no genomic DNA contamination
Sample Type Biological Fluids, Cell Culture Cells, Tissue Samples
Specificity The Mitochondrial DNA Extraction Kit provides convenient tools for isolating mtDNA from a variety of cells and tissues in high yield and purity, without contaminations from genomic DNA. The purified mtDNA can be used for a variety of studies such as enzyme manipulations, Southern blotting, cloning, PCR analysis, and amplifications.
Characteristics Mitochondrial DNA Isolation Kit: Rapid & Convenient Kit for Isolating mtDNA from a variety of Cells and Tissues. High yield and Purity. No genomic DNA contamination.
Mitochondria are semiautonomous organelles which functions in aging process, apoptosis, anti-HIV drugs, and cancers. Mitochondrial DNA (mtDNA) has a very high mutation rate and the mutations on mtDNA appear to be related to certain diseases such as diabetes, Alzheimer's disease, and muscle disorders. Isolation and quantification of mtDNA are often required to study the relationships between the diseases and mtDNA. The Mitochondrial DNA Extraction Kit provides convenient tools for isolating mtDNA from a variety of cells and tissues in high yield and purity, without contaminations from genomic DNA. The purified mtDNA can be used for a variety of studies such as enzyme manipulations, Southern blotting, cloning, PCR analysis, and amplifications.
Components
  • 5X Cytosol Extraction Buffer
  • Mitochondrial Lysis Buffer
  • Enzyme B Mix (lyophilized)
  • TE Buffer
Application Notes Applications: Enzyme manipulations, Southern blotting, cloning, PCR analysis, and amplifications.
Comment


Simple procedure, takes less than 2 hours
Fast and convenient
The kit provides unique formulations of buffers and reagents for isolating a highly enriched mitochondrial fraction. Mitochondrial DNA are then isolated using reagents included in the kit

Protocol Note: To check the efficiency of homogenization, pipette 2-3 µL of the homogenized suspension onto a coverslip and observe under a microscope. A shiny ring around the nuclei indicates that cells are still intact. If 70-80 % of the nuclei do not have the shiny ring, proceed to
6. Otherwise, perform 30-50 additional passes using the dounce tissue grinder. Excessive homogenization should also be avoided, as it can cause damage to the mitochondrial membrane which triggers release of mitochondrial components.
6. Transfer homogenate to a 1.5-ml microcentrifuge tube, and centrifuge at 700 x g for 10 minutes at 4 °C. The step removes nuclei and intact cells (in pellet).
7. Transfer supernatant to a fresh 1.5-ml tube, and centrifuge at 10,000 x g for 30 minutes at 4 °C.
8. Remove supernatant.
9. Resuspend the pellet in 1 mL 1X Cytosol Extraction Buffer and centrifuge at 10000 x g for 30 minutes at 4 °C again. 10. Remove the supernatant. The pellet is isolated mitochondria. 11. Lyse the mitochondria in 30 µL of the Mitochondrial Lysis Buffer, keep on ice for 10 minutes. 12. Add 5 µL Enzyme B Mix and incubate at 50 °C water bath for 60 min or longer until the solution becomes clear. 13. Add 100 µL absolute ethanol, mix and keep at -20 °C for 10 minutes. 14. Centrifuge in microcentrifuge at top speed for 5 min at room temperature. 15. Remove the supernatant. The pellet is mitochondrial DNA. 16. Wash the DNA pellet 2 times with 1 mL of 70 % ethanol. Remove the trace amount ethanol using pipet tip. Air dry for 5 min. (Do not completely dry the DNA. It may be difficult to dissolve if it is completely dried.) 17. Resuspend the DNA in 20 µL TE buffer or water. Store the extracted DNA at -20 °C for future use. (Generally, 5-20 µg mtDNA can be generated for each isolation.)
Restrictions For Research Use only
Storage -20 °C
Expiry Date 12 months
Product cited in: Imbaby, Ewais, Essawy, Farag: "Cardioprotective effects of curcumin and nebivolol against doxorubicin-induced cardiac toxicity in rats." in: Human & experimental toxicology, Vol. 33, Issue 8, pp. 800-13, 2015 (PubMed).

Xiang, Lan, Tang, Yuan, Xu, Zhao, Li, Zhang: "Tuberous sclerosis complex 1-mechanistic target of rapamycin complex 1 signaling determines brown-to-white adipocyte phenotypic switch." in: Diabetes, Vol. 64, Issue 2, pp. 519-28, 2015 (PubMed).

García-Ruiz, Solís-Muñoz, Fernández-Moreira, Muñoz-Yagüe, Solís-Herruzo: "In vitro treatment of HepG2 cells with saturated fatty acids reproduces mitochondrial dysfunction found in nonalcoholic steatohepatitis." in: Disease models & mechanisms, Vol. 8, Issue 2, pp. 183-91, 2015 (PubMed).

Torres-Gonzalez, Gawlowski, Kocalis, Scott, Dillmann: "Mitochondrial 8-oxoguanine glycosylase decreases mitochondrial fragmentation and improves mitochondrial function in H9C2 cells under oxidative stress conditions." in: American journal of physiology. Cell physiology, Vol. 306, Issue 3, pp. C221-9, 2014 (PubMed).

Zhang, Konstantinidis, Yang, Mizukawa, Kalim, Lang, Kalfa, Zheng, Guo: "Gene targeting RhoA reveals its essential role in coordinating mitochondrial function and thymocyte development." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 193, Issue 12, pp. 5973-82, 2014 (PubMed).

Mishra, Raghuram, Jain, Jain, Khare, Pathak: "Mitochondrial oxidative stress-induced epigenetic modifications in pancreatic epithelial cells." in: International journal of toxicology, Vol. 33, Issue 2, pp. 116-29, 2014 (PubMed).

Ries, Schuster, Thomann, Donhauser, Vollmer, Schmidt: "Identification of novel oligonucleotides from mitochondrial DNA that spontaneously induce plasmacytoid dendritic cell activation." in: Journal of leukocyte biology, Vol. 94, Issue 1, pp. 123-35, 2013 (PubMed).

Tomoeda, Yuki, Kubo, Yoshizawa, Kitamura, Nagata, Nishizawa, Tomita: "Role of Meis1 in mitochondrial gene transcription of pancreatic cancer cells." in: Biochemical and biophysical research communications, Vol. 410, Issue 4, pp. 798-802, 2011 (PubMed).