Mitochondria are double-membrane cytoplasmic organelles that generate over 90% of a cell’s chemical energy through respiration. The mitochondrial genome (mtDNA) is a circular, intron-free, double-stranded, haploid DNA molecule approximately 16.5 kb in length that encodes 37 genes. Unlike nuclear DNA, mtDNA exhibits a high rate of mutation, damage, and copy number variation, which has been closely linked to a variety of diseases, including diabetes, Alzheimer’s disease, and muscle disorders. To investigate these associations, it is essential to obtain high-quality mtDNA.

The RayBio^® Mitochondrial DNA Isolation Kit (Column-Based) provides a simple and efficient method for isolating mtDNA (1 hour for 12 samples) from a variety of cells. The use of a spin column allows for higher sensitivity over bead-based isolation methods. This kit yields highly pure mtDNA, free of nuclear genomic DNA contamination, and is suitable for a wide range of downstream applications, including enzymatic manipulation, Southern blotting, cloning, PCR analysis, and amplification.

• Simple, efficient and higher sensitivity
• Isolates high-quality mtDNA
• No nuclear DNA contamination
• High yield and purity
• Isolates mtDNA in 1 hour
• Easy to use

• Sterile nuclease-free pipette tips (barrier tips recommended) and microfuge tubes
• PBS
• Microcentrifuge
• Vortex Mixer
• Water bath or dry heat block capable of heating to 56°C
• Precision pipettes to deliver 2 µL to 1 mL volumes
• Tissue homogenizer if using tissue samples (Dounce tissue grinder recommended)
• Refrigerated centrifuge with swing out rotor and refrigerated microcentrifuge

Contact our technical support team for questions about compatibility: techsupport@raybiotech.com

1. Prepare cell or tissue samples as instructed in the manual.
2. Lyse cells with Cell Lysis Buffer 1 for 15 min and Cell Lysis Buffer 2 for another 2 minutes on ice.
3. Lyse mitochondria with Protease K and mtDNA Lysis Buffer at 56°C for 10 minutes.
4. Add Binding Buffer 2, vortex, transfer to a spin column, and centrifuge.
5. Wash with Wash Buffers 1 and 2.
6. Elute mtDNA with Elution Buffer.
7. mtDNA can be used immediately or stored at or below -20°C.

Store Cell Lysis Buffer and Protease K at 4°C. Store the rest of the components in a dry environment between 2 - 30°C. The kit can be stored for up to 12 months.