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| Product Name | Silica-Membrane Plasmid Miniprep Kit |
| Catalog No. | NATR-HMM-0109 |
| Description | A high-throughput compatible silica membrane spin column kit for the rapid purification of plasmid DNA from Escherichia coli cultures. The alkaline lysis method combined with silica membrane purification delivers high-copy plasmid DNA suitable for transfection, sequencing, cloning, and other molecular biology applications. |
| Intended Use | Isolation of plasmid DNA from 1–5 mL overnight E. coli cultures for routine molecular cloning, restriction enzyme analysis, PCR template preparation, and Sanger sequencing. |
| Principle / Technology | Alkaline lysis with SDS and NaOH disrupts bacterial cells and denatures chromosomal DNA and proteins while leaving plasmid DNA intact. After neutralization with acetate buffer, plasmid DNA renatures while genomic DNA and proteins precipitate. The cleared lysate passes through a silica membrane in the presence of chaotropic salts; washes remove residual contaminants; purified plasmid DNA is eluted in low-ionic-strength buffer. |
| Detection Method | UV spectrophotometry (A260/A280 and A260/A230 ratios); agarose gel electrophoresis for supercoiled/nicked/linear confirmation; restriction digestion for insert verification; fluorometric quantitation |
| Sample Type | E. coli cultures (DH5α, TOP10, XL1-Blue, JM109, and other standard cloning strains) grown in LB or 2xYT medium; compatible with high-copy and low-copy plasmids |
| Performance Range / Specifications | Typical yield from 3 mL overnight culture: 10–25 μg (high-copy plasmid), 3–8 μg (low-copy plasmid); A260/A280 ratio: 1.80–1.95; endotoxin: 0.1–1.0 EU/μg for standard protocol |
| Sensitivity / LOD | Recovers plasmid DNA from cultures with OD600 as low as 0.5; minimum detectable plasmid yield: 200 ng |
| Specificity | Purified plasmid DNA is free of genomic DNA, RNA, protein, and bacterial endotoxin; no detectable nucleases; suitable for enzymatic manipulation |
| Reaction Conditions / Protocol | Pellet 1–5 mL overnight culture, resuspend in 250 μL resuspension buffer (with RNase A), add 250 μL lysis buffer, mix by gentle inversion, incubate ≤5 minutes, add 350 μL neutralization buffer, mix, centrifuge 14,000g for 10 minutes, transfer supernatant to spin column, centrifuge 1 minute, wash with 500 μL wash buffer, wash with 700 μL wash buffer, dry spin, elute with 30–100 μL elution buffer or water; total time approximately 30 minutes |
| Components / Formulation | Resuspension buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 100 μg/mL RNase A), lysis buffer (200 mM NaOH, 1% SDS), neutralization buffer (3 M potassium acetate pH 5.5), wash buffer concentrate (Tris-HCl, NaCl, EDTA, ethanol), elution buffer (10 mM Tris-HCl pH 8.5), silica membrane spin columns with collection tubes |
| Storage Conditions | RNase A-containing resuspension buffer at 2–8°C; all other buffers at room temperature; lyophilized RNase A at 2–8°C before reconstitution |
| Shelf Life | 24 months from date of manufacture; resuspension buffer with RNase A stable for 6 months at 2–8°C after preparation |
| Package Specifications | 50, 100, and 250 preparation kits; all necessary plasticware included |
| Product Form | Liquid buffers; silica membrane spin columns; lyophilized RNase A |
| Quality Control | Each lot tested with pUC19 control plasmid: yield ≥15 μg from 3 mL DH5α culture; A260/A280: 1.80–1.95; >90% supercoiled form; functional testing by EcoRI restriction digestion and Sanger sequencing; endotoxin quantified by LAL assay |
| Key Features | Streamlined alkaline lysis protocol; endotoxin levels suitable for standard transfection; RNase A pre-added to resuspension buffer for convenience; high-purity DNA compatible with automated fluorescent sequencing |
For research use only, not for clinical use.
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