Mitochondrial DNA Isolation Kit

Application: Immunoprecipitation (IP)

Catalog no.: ABIN412491

Additional Information

Description: 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.

Application Details

Application Notes: Note: To check the efficiency of homogenization, pipette 2-3 μ l 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 step 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 o 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 o 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 o C again. 10. Remove the supernatant. The pellet is isolated mitochondria. 11. Lyse the mitochondria in 30 μ l of the Mitochondrial Lysis Buffer, keep on ice for 10 minutes. 12. Add 5 μ l Enzyme B Mix and incubate at 50 o C water bath for 60 min or longer until the solution becomes clear. 13. Add 100 μ l absolute ethanol, mix and keep at –20 o 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 μ l TE buffer or water. Store the extracted DNA at –20 o C for future use. (Generally, 5-20 μ g mtDNA can be generated for each isolation.)

Restrictions: For Research Use only

Publications

Publications: Tomoeda, Yuki, Kubo et al.: "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).