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| Product Name | Column-Based Gel Extraction and PCR Cleanup Kit |
| Catalog No. | DREK-0015 |
| Description | A dual-purpose silica membrane spin column kit for the purification of DNA fragments from agarose gels and the direct cleanup of PCR amplification products. The optimized binding buffer solubilizes agarose gels at moderate temperature and provides precise pH-controlled conditions for selective binding of DNA fragments while excluding primers, primer-dimers, enzymes, unincorporated nucleotides, and salts. The kit is effective across a broad fragment size range, from oligonucleotides to large PCR products. |
| Intended Use | Purification of DNA fragments from agarose gels following electrophoresis for subcloning, probe preparation, and sequencing; cleanup of DNA from PCR and other enzymatic reactions to remove primers, dNTPs, enzymes, and buffer salts for downstream applications. |
| Principle / Technology | Agarose gel slices containing DNA fragments are dissolved in a chaotropic salt-based solubilization buffer at 50-55°C. The dissolved agarose solution is applied to a silica membrane spin column where DNA selectively binds under high-salt, mildly acidic conditions. Primers, dNTPs, and other small molecules pass through the column. After ethanolic wash steps, purified DNA is eluted in a low-salt buffer or water. For PCR cleanup, the amplification reaction is mixed directly with binding buffer and processed through the same column protocol. |
| Detection Method | Agarose gel electrophoresis for size confirmation and purity; UV spectrophotometry for concentration and purity; fluorometric quantitation for precise yield; Sanger sequencing for fragment accuracy; restriction digestion for functional validation |
| Sample Type | DNA fragments in agarose gels (TAE or TBE buffer, standard or low-melting-point agarose up to 3%); PCR amplification products from Taq, proofreading, and high-fidelity DNA polymerases; restriction enzyme digests; labeling reactions; DNA from ligation and end-repair reactions |
| Performance Range / Specifications | Fragment size range: 70 bp to 10 kb; gel slice mass: up to 400 mg per column; binding capacity: 10 µg DNA per column; recovery efficiency: 70-95% (size-dependent: >90% for 100 bp-5 kb, >70% for 50-100 bp and 5-10 kb); elution volume: 15-50 µL; A260/A280 of purified DNA: 1.80-1.95; primer removal: >99% |
| Sensitivity / LOD | Recovers DNA fragments as small as 40 bp; visual detection of <50 ng DNA on agarose gel post-purification; effective purification from gel slices containing <100 ng input DNA; consistent recovery across TAE and TBE gel systems |
| Specificity | Purified DNA fragments free of agarose, ethidium bromide/SYBR Safe, enzymes, and buffer salts; no detectable DNA polymerase activity; complete removal of primers and primer-dimers (invisible on analytical gel); ligation-compatible DNA (blunt-end and cohesive-end cloning efficiency equivalent to column-only purified DNA) |
| Reaction Conditions / Protocol | For gel extraction: excise DNA band with clean scalpel under UV transillumination (minimize exposure); weigh gel slice; add 3 volumes solubilization buffer per gel mass (300 µL per 100 mg); incubate at 50-55°C for 10 minutes with vortexing every 2-3 minutes until completely dissolved; add 1 gel volume isopropanol; transfer to spin column; centrifuge at 13,000 rpm for 1 minute; wash with 500 µL wash buffer; centrifuge; wash with 700 µL wash buffer; centrifuge; dry spin 2 minutes; elute with 15-50 µL elution buffer or water; incubate 2 minutes; centrifuge; total time: 20-30 minutes. For PCR cleanup: add 5 volumes binding buffer to PCR reaction, mix, transfer to column, centrifuge, wash, elute as above. |
| Components / Formulation | Solubilization/binding buffer (sodium iodide or guanidine hydrochloride, Tris-HCl, sodium acetate, pH indicator dye), wash buffer concentrate (Tris-HCl, NaCl, EDTA, ethanol), elution buffer (10 mM Tris-HCl pH 8.5, 0.1 mM EDTA), silica membrane spin columns with collection tubes; isopropanol required but not supplied |
| Storage Conditions | All buffers and spin columns at room temperature (15-25°C); protect binding buffer from light; keep buffer bottles tightly closed to prevent evaporation; no special storage requirements |
| Shelf Life | 24 months from date of manufacture; buffers stable for 12 months after opening; columns stable indefinitely in sealed pouch |
| Package Specifications | 50 preparations and 250 preparations; includes all buffers and spin columns; isopropanol not included |
| Product Form | Silica membrane spin columns with collection tubes; liquid buffers with pH indicator for visual confirmation of correct binding pH |
| Quality Control | Each lot validated with DNA ladder fragments: recovery >85% for 500 bp fragment, >75% for 5 kb fragment, >65% for 50 bp and 8 kb fragments; PCR cleanup: >99% primer removal; A260/A280: 1.80-1.95; ligation efficiency >95% relative to untreated DNA; no DNase activity |
| Key Features | Dual-function kit for gel extraction and PCR cleanup; color indicator confirms optimal binding pH; high recovery across broad fragment range; 20-minute protocol; eluted DNA ready for all enzymatic reactions; no phenol or organic extractions; room temperature storage |
| Purity | A260/A280: 1.80-1.95; A260/A230: >1.8; agarose, salts, and primer contamination below detection limits |
| Concentration | Elution volume 15-50 µL; DNA concentration typically 10-200 ng/µL dependent on input amount and fragment size |
| Activity / Unit Definition | Not applicable for purification kits |
| Molecular Weight | Not applicable; purifies DNA fragments across a broad molecular weight range |
| Source / Origin | Silica membrane from quartz glass microfiber; binding buffer based on chaotropic salt chemistry (sodium iodide or guanidine hydrochloride); all buffers are synthetic, nuclease-free certified |
| pH Range / Optimal pH | Binding buffer pH 5.0-6.0 (optimal DNA binding to silica); wash buffer pH 7.0-7.5; elution buffer pH 8.3-8.7 |
| Shipping Conditions | Ambient temperature shipping; no special handling or dangerous goods classification required; stable in transit for extended periods |
| Expiration Date / Stability | 24 months shelf life; real-time stability confirmed at 24 months including recovery efficiency and DNA quality metrics; binding buffer pH verified stable throughout shelf life; accelerated aging at 45°C for 28 days shows maintained performance |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic applications; manufactured in ISO 9001 certified facility; certificate of analysis with lot-specific recovery data |
| Compatibility | Purified DNA suitable for all standard molecular biology applications: restriction digestion, ligation, transformation, Sanger sequencing, NGS library preparation, PCR, qPCR, in vitro transcription, labeling reactions, and cloning; compatible with DNA from TAE and TBE gel systems; works with ethidium bromide, SYBR Safe, GelRed, and other common DNA stains |
| Recommended Buffer System | Chaotropic salt-based solubilization and binding buffer; ethanol-based wash buffer; Tris-EDTA elution buffer; pH indicator dye system integrated |
| Application Notes / Precautions | Minimize UV exposure time during gel excision to prevent DNA damage; for UV-sensitive applications, use blue-light transilluminator; ensure complete gel dissolution before loading column; if binding buffer turns from yellow to violet/purple, add sodium acetate to restore correct pH; for fragments <100 bp, add isopropanol at 1.5× gel volume; for fragments >5 kb, preheat elution buffer to 50°C; for low-yield fragments, reduce elution volume and perform second elution with fresh buffer; avoid over-drying column membrane |
| Batch-to-Batch Consistency | Between-lot DNA recovery CV <10%; binding buffer pH within ±0.2 units between lots; column binding capacity ≥10 µg verified per lot using λ DNA HindIII ladder; primer removal efficiency >99% verified per lot by qPCR; DNase contamination testing per batch |
For research use only, not for clinical use.
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