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| Product Name | Column-Based Plasmid DNA Mini Extraction Kit |
| Catalog No. | DREK-0014 |
| Description | An alkaline lysis-based silica membrane spin column kit for the rapid purification of high-quality plasmid DNA from Escherichia coli cultures. The optimized protocol yields up to 20 µg of high-copy plasmid DNA suitable for transfection, cloning, sequencing, and other molecular biology applications. The kit provides consistent results across common cloning strains with yields proportional to plasmid copy number and culture density. |
| Intended Use | Isolation of plasmid DNA from 1-5 mL E. coli overnight cultures for standard molecular cloning, restriction enzyme digestion, PCR template preparation, fluorescent and Sanger sequencing, site-directed mutagenesis, and in vitro transcription. |
| Principle / Technology | Bacterial cells are harvested and lysed under alkaline conditions with SDS and NaOH, which disrupts the cell wall, denatures chromosomal DNA and proteins, and releases plasmid DNA. The solution is neutralized with potassium acetate, causing genomic DNA and proteins to precipitate while plasmid DNA renatures. The cleared lysate is passed through a silica membrane column in the presence of a high-salt binding buffer, where plasmid DNA binds. After washing, purified plasmid DNA is eluted in a low-ionic-strength buffer or water. |
| Detection Method | UV spectrophotometry (A260/A280); agarose gel electrophoresis for supercoiled/nicked/linear plasmid forms; restriction endonuclease digestion for insert verification; Sanger sequencing for insert accuracy; transfection efficiency in HEK293 or CHO cells |
| Sample Type | E. coli cultures (DH5α, TOP10, JM109, XL1-Blue, Stbl3, and other common cloning strains) grown in LB, 2xYT, TB, or SOC medium; compatible with high-copy (pUC origin, >300 copies/cell), medium-copy (pBR322 origin, 15-20 copies/cell), and low-copy plasmids |
| Performance Range / Specifications | Input: 1-5 mL overnight culture (OD600 2.0-4.0); yield from 3 mL DH5α/pUC19: 10-25 µg (high-copy); from 5 mL DH5α/pBR322: 3-8 µg (low-copy); A260/A280: 1.80-1.95; A260/A230: >1.8; column binding capacity: 25 µg; >90% supercoiled form; endotoxin: 0.5-5 EU/µg |
| Sensitivity / LOD | Consistent plasmid DNA recovery from cultures with OD600 as low as 0.5; successful sequencing reads from as little as 50 ng template; effectively recovers plasmids up to 15 kb; low-copy plasmid yields sufficient for sequencing applications |
| Specificity | Purified plasmid DNA free of genomic DNA, RNA, and protein; endotoxin levels suitable for standard transfection applications; no detectable RNase or DNase activity in eluate; restriction digestion produces expected fragment patterns |
| Reaction Conditions / Protocol | Pellet 1-5 mL overnight culture at 13,000 rpm (or equivalent g-force) for 30 seconds; resuspend in 250 µL resuspension buffer with RNase A; add 250 µL lysis buffer; mix by gentle inversion 4-6 times; incubate <5 minutes until lysate clears; add 350 µL neutralization buffer; mix immediately by inversion; centrifuge at 13,000 rpm for 10 minutes; transfer supernatant to spin column; centrifuge 30-60 seconds; wash with 500 µL wash buffer; centrifuge; wash with 700 µL wash buffer; centrifuge; dry spin 1 minute; elute with 30-50 µL elution buffer or water; incubate 1 minute; centrifuge 1 minute; total time: 20-30 minutes for 1-24 samples |
| 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, guanidine hydrochloride), 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 |
| Storage Conditions | Resuspension buffer with RNase A at 2-8°C; lysis buffer and neutralization buffer at room temperature; wash buffer at room temperature; elution buffer at room temperature; columns at room temperature; check resuspension buffer for RNase A activity periodically |
| Shelf Life | 24 months from date of manufacture; resuspension buffer with RNase A stable for 6 months at 2-8°C after preparation; lysis buffer stable for 12 months after opening if SDS remains in solution |
| Package Specifications | 50 preparations, 100 preparations, and 250 preparations; includes all columns, collection tubes, and reagents |
| Product Form | Silica membrane spin columns with collection tubes; liquid buffers; RNase A pre-added to resuspension buffer (lyophilized RNase A reconstituted) |
| Quality Control | Each lot tested with pUC19 in DH5α: yield ≥15 µg from 3 mL culture; A260/A280: 1.80-1.95; >90% supercoiled form by agarose gel; EcoRI digestion complete; Sanger sequencing >800 bp read length with >98% accuracy; endotoxin <5 EU/µg; RNase/DNase free; genomic DNA free by PCR |
| Key Features | Fast 20-minute alkaline lysis protocol; RNase A pre-integrated; consistent yields across standard cloning strains; no phenol-chloroform extractions; endotoxin levels suitable for routine transfection; vacuum manifold compatible for high-throughput |
| Purity | A260/A280: 1.80-1.95; A260/A230: >1.8; gDNA contamination undetectable by qPCR; protein <0.1% by Bradford |
| Concentration | Elution volume 30-50 µL; plasmid DNA concentration 200-500 ng/µL for high-copy plasmids from 3 mL culture; lower for low-copy plasmids |
| Activity / Unit Definition | Not applicable for plasmid DNA extraction kits |
| Molecular Weight | Not applicable; plasmid molecular weight depends on construct size (typically 2-15 kb, corresponding to ~1.3-10 MDa for double-stranded circular DNA) |
| Source / Origin | Silica membrane made from high-purity glass microfiber; RNase A from bovine pancreas; all buffer components are synthetic, analytical grade |
| pH Range / Optimal pH | Resuspension buffer pH 7.8-8.2; lysis buffer pH 12.0-12.5; neutralization buffer pH 5.3-5.7; wash buffer pH 7.0-7.5; elution buffer pH 8.3-8.7 |
| Shipping Conditions | Ambient temperature for all components; resuspension buffer with RNase A shipped with cold packs to maintain enzyme activity during transit; standard courier service acceptable |
| Expiration Date / Stability | 24 months for dry components; resuspension buffer with RNase A: 6 months at 2-8°C; accelerated stability: lysis and neutralization buffers stable at 40°C for 30 days; wash buffer ethanol content verified throughout shelf life |
| Regulatory / Compliance | For research use only; not for in vivo therapeutic applications without additional endotoxin removal; manufactured in ISO 9001 facility; certificate of analysis available per lot |
| Compatibility | Plasmid DNA suitable for restriction digestion, ligation, transformation, PCR, Sanger and next-generation sequencing, in vitro transcription, and standard transfection; compatible with vacuum manifold processing for 96-well format when using plate adapters |
| Recommended Buffer System | Tris-EDTA resuspension; NaOH-SDS alkaline lysis; potassium acetate-guanidine hydrochloride neutralization; silica membrane binding; Tris-EDTA elution |
| Application Notes / Precautions | Do not vortex after lysis step to avoid shearing genomic DNA; ensure complete neutralization (solution should become cloudy with white precipitate); do not exceed 5 mL culture for high-copy plasmids to avoid column overloading; for low-copy plasmids, increase culture volume to 10-15 mL and process in aliquots; if protein pellet is loose after centrifugation, recentrifuge or filter lysate; pre-warm elution buffer for maximum yield |
| Batch-to-Batch Consistency | DNA yield between lots CV <10% for standardized E. coli lysate; A260/A280 variation <0.03; column binding capacity maintained within ±10% specification; RNase A activity verified >5 Kunitz units/µL; endotoxin level monitored per lot |
For research use only, not for clinical use.
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