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| Product Name | Mitochondrial DNA Isolation Kit, Spin Column Method, Cells and Tissues |
| Catalog No. | DREK-0030 |
| Description | Spin column-based kit designed for rapid and specific isolation of mitochondrial DNA (mtDNA) from cultured cells and fresh tissue samples without nuclear DNA contamination. The protocol employs a differential centrifugation step to enrich mitochondria followed by alkaline lysis of the mitochondrial pellet and silica membrane column purification of the released mtDNA. The resulting mtDNA is free of nuclear genomic DNA, RNA, and protein contaminants and is suitable for mtDNA copy number quantification, heteroplasmy analysis, mitochondrial genome sequencing, and mutation screening. The kit incorporates an optional DNase I treatment step to eliminate residual nuclear DNA from the mitochondrial fraction prior to lysis. |
| Intended Use | Isolation of highly purified mitochondrial DNA from cultured mammalian cells (minimum 1x10^7 cells), liver, heart, brain, muscle, and kidney tissue for mitochondrial genome studies including mutation analysis, heteroplasmy detection, copy number quantification by qPCR, and next-generation sequencing library preparation. |
| Principle / Technology | Differential centrifugation (600 x g remove nuclei, 10,000 x g pellet mitochondria); optional DNase I treatment of mitochondrial fraction to remove contaminating nuclear DNA; alkaline lysis of mitochondria; binding of mtDNA to silica membrane in high-salt buffer; wash steps remove proteins and salts; elution in low-ionic-strength buffer or water. |
| Detection Method | Homogenize cells/tissue in isotonic mitochondrial isolation buffer; centrifuge 600 x g, 10 min, 4 C (pellet = nuclei/debris); transfer supernatant; centrifuge 10,000 x g, 15 min, 4 C (pellet = mitochondria); optional: resuspend pellet in DNase I buffer, incubate 20 min RT; wash mitochondrial pellet; lyse in alkaline lysis buffer; neutralize; bind mtDNA to column; wash 2x; elute in 50-100 uL elution buffer. |
| Sample Type | Fresh cultured cells (1x10^7 to 5x10^7 cells), fresh liver (50-100 mg), heart (50-100 mg), brain (50-100 mg), skeletal muscle (50-100 mg), kidney (50-100 mg). Not recommended for frozen or fixed samples. |
| Performance Range / Specifications | mtDNA yield: 0.5-5 ug per preparation depending on cell/tissue type and mitochondrial content; mtDNA purity: >95% mitochondrial DNA (no nuclear 18S rRNA gene detected by qPCR); A260/A280 ratio 1.7-1.9; mtDNA fragment size >16 kb (intact circular mtDNA). |
| Sensitivity / LOD | Recovery of mtDNA from as few as 1x10^6 cells (HeLa); detectable by qPCR targeting mitochondrial D-loop region; detection limit approximately 100 copies mtDNA by qPCR. |
| Specificity | Selective enrichment of mitochondria by differential centrifugation; DNase I treatment eliminates nuclear DNA contamination; silica membrane binds double-stranded DNA (both mtDNA and any residual nDNA); mitochondrial origin verified by absence of nuclear gene amplification (GAPDH, beta-actin) and presence of mitochondrial genes (COX1, ND1). |
| Reaction Conditions / Protocol | Total protocol time 2-3 hours from sample to purified mtDNA; all centrifugation steps at 4 C; DNase I incubation 20 min at RT; column binding and washing at RT; elution with pre-warmed (60 C) buffer for higher yield. |
| Components / Formulation | Mitochondrial Isolation Buffer (5x concentrate), Mitochondrial Wash Buffer (10x), DNase I (RNase-free, lyophilized), DNase I Reaction Buffer (10x), Alkaline Lysis Buffer, Neutralization Buffer, Binding Buffer, Wash Buffer (concentrate), Elution Buffer (10 mM Tris-HCl, pH 8.5), Spin Columns with Collection Tubes. |
| Storage Conditions | Mitochondrial Isolation Buffer and Wash Buffer at 2-8 C; DNase I at -20 C; all other components at RT (15-25 C); protect DNase I from moisture. |
| Shelf Life | 12 months from date of manufacture. |
| Package Specifications | 20 preparations, 50 preparations. |
| Product Form | Liquid buffers; lyophilized DNase I; silica membrane spin columns. |
| Quality Control | Each lot tested for mtDNA yield from HeLa cells and rat liver tissue; purity verified by qPCR for nuclear (18S rDNA) and mitochondrial (MT-COX1) markers (nuclear DNA less than 5% of total); DNase I activity 2,000 units/mg. |
| Key Features | Specific mtDNA isolation with no nuclear DNA contamination; optional DNase I step for maximum purity; spin column format for simplicity; suitable for sequencing and heteroplasmy analysis; ~2-3 hour protocol. |
| Purity | mtDNA purity >95% by qPCR; A260/A280 1.7-1.9; no detectable RNase or DNase contamination in eluate. |
| Concentration | Typical yield 1-5 ug mtDNA per 50 mg tissue; concentration in 50-100 uL elution volume: 10-100 ng/uL. |
| Activity / Unit Definition | DNase I: specific activity >=2,000 Kunitz units/mg protein. |
| Molecular Weight | mtDNA size approximately 16,569 bp (human); DNase I approximately 31 kDa. |
| Source / Origin | Kit components synthetic and recombinant origin; silica membrane columns manufactured in ISO 13485 certified facility. |
| pH Range / Optimal pH | Mitochondrial isolation buffer pH 7.4; alkaline lysis pH 12.0-12.5; binding buffer pH 5.0-6.0; elution buffer pH 8.5. |
| Shipping Conditions | Ambient temperature (RT) for buffers and columns; DNase I on blue ice (-20 C). |
| Expiration Date / Stability | 12 months at recommended storage conditions; DNase I stable at -20 C for 12 months; avoid freeze-thaw cycles of DNase I and buffers. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use; manufactured under ISO 9001 quality management system. |
| Compatibility | Compatible with downstream qPCR, Sanger sequencing, NGS library preparation (after mtDNA quantification), restriction enzyme digestion, and long-range PCR of mtDNA. Elution buffer is EDTA-free for compatibility with magnesium-dependent enzymatic reactions. |
| Recommended Buffer System | Mitochondrial isolation: sucrose-mannitol-HEPES pH 7.4; alkaline lysis: NaOH-SDS pH 12-12.5; binding: guanidine HCl pH 5-6; elution: 10 mM Tris-HCl pH 8.5. |
| Application Notes / Precautions | Mitochondrial DNA copy number varies dramatically between cell types (100-10,000 copies per cell). Tissues with high mitochondrial density (heart, skeletal muscle, brown adipose) yield more mtDNA. Keep samples cold and process fresh whenever possible. Avoid vortexing mitochondrial pellets to prevent damage. DNase I treatment is recommended but optional; if nuclear DNA contamination is acceptable for downstream applications, skip DNase step to increase mtDNA yield. mtDNA is susceptible to oxidative damage during isolation; work quickly and avoid exposure to UV light. |
| Batch-to-Batch Consistency | mtDNA yield within +/-20% of reference lot; nuclear DNA contamination <5% by qPCR for every lot; DNase I activity within +/-15%. |
For research use only, not for clinical use.
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