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| Product Name | Bacterial Plasmid DNA Midiprep Kit, Gravity-Flow Column, Transfection Grade |
| Catalog No. | DREK-0031 |
| Description | Gravity-flow anion-exchange column kit for preparative-scale purification of transfection-grade plasmid DNA from recombinant E. coli cultures (25-100 mL overnight culture). The method uses modified alkaline lysis followed by anion-exchange chromatography under low-pressure gravity flow to separate supercoiled plasmid DNA from RNA, chromosomal DNA, proteins, and endotoxins based on charge density differences. The final ethanol-precipitated plasmid DNA is of transfection quality with endotoxin levels below 0.1 EU/ug and is suitable for mammalian cell transfection, microinjection, in vivo gene delivery, and other applications requiring high-purity supercoiled plasmid. |
| Intended Use | Purification of transfection-grade ultra-pure supercoiled plasmid DNA from 25-100 mL E. coli cultures for mammalian cell transfection (lipid-based, electroporation), in vivo gene therapy research, microinjection, biolistic delivery, and lentiviral/AAV packaging plasmid preparation. |
| Principle / Technology | Modified alkaline lysis (NaOH/SDS) releases plasmid DNA in supercoiled form while denaturing genomic DNA and proteins; neutralization with potassium acetate precipitates genomic DNA, proteins, and SDS as a white flocculent; cleared lysate loaded onto anion-exchange column where plasmid DNA binds (negatively charged phosphate backbone interacts with positively charged DEAE groups); sequential wash steps remove RNA and residual contaminants; high-salt elution releases plasmid DNA; isopropanol precipitation concentrates and desalts plasmid DNA. |
| Detection Method | Harvest 25-100 mL overnight bacterial culture by centrifugation; resuspend in Buffer P1 (Tris-EDTA + RNase A); add Buffer P2 (NaOH/SDS), mix gently, incubate <=5 min; add Buffer P3 (potassium acetate), mix, incubate on ice 15 min; centrifuge >=15,000 x g, 30 min, 4 C; load supernatant onto equilibrated anion-exchange column by gravity flow; wash with Buffer QC (medium salt); elute plasmid DNA with Buffer QF (high salt); precipitate with 0.7 volumes isopropanol; centrifuge >=15,000 x g, 30 min, 4 C; wash pellet with 70% ethanol; air-dry; resuspend in TE or water. |
| Sample Type | 25-100 mL overnight E. coli culture harboring high-copy or low-copy plasmid; recommended cell density OD600 3-6. |
| Performance Range / Specifications | Plasmid yield: 100-500 ug per preparation from 25-100 mL high-copy plasmid culture; A260/A280 ratio 1.80-1.95; endotoxin <0.1 EU/ug DNA; >90% supercoiled plasmid (covalently closed circular form) as assessed by agarose gel electrophoresis. |
| Sensitivity / LOD | Minimum detectable plasmid by spectrophotometry: approximately 250 ng/mL (0.005 A260 units); agarose gel detection limit approximately 10-50 ng per band. |
| Specificity | Anion-exchange chromatography selectively binds nucleic acids (plasmid DNA, residual RNA) based on phosphate backbone charge density; sequential salt elution separates RNA (elutes at lower salt) from plasmid DNA (elutes at higher salt); >90% of recovered DNA is supercoiled plasmid form. |
| Reaction Conditions / Protocol | Total protocol time approximately 3-4 hours (plus organism culture time); alkaline lysis incubation <=5 min (exceeding may denature supercoiled plasmid); isopropanol precipitation 30 min centrifugation at 4 C; optional endotoxin removal column extension adds 30 min. |
| Components / Formulation | Buffer P1 (Resuspension Buffer with RNase A), Buffer P2 (Lysis Buffer), Buffer P3 (Neutralization Buffer), Buffer QC (Wash Buffer), Buffer QF (Elution Buffer), Anion-Exchange Columns (pre-packed), Column Adapters and Syringe Connectors, Protocol. |
| Storage Conditions | Buffer P1 at 2-8 C after adding RNase A; RNase A stock at -20 C; all other buffers and columns at RT (15-25 C); protect Buffer P2 from air (keep tightly closed). |
| Shelf Life | Buffers and columns: 18 months from date of manufacture; RNase A: 24 months at -20 C. |
| Package Specifications | 10 preparations, 25 preparations. |
| Product Form | Liquid buffers; pre-packed gravity-flow columns; lyophilized RNase A. |
| Quality Control | Each lot tested for yield from pUC19-transformed DH5alpha (>=200 ug per 50 mL culture); endotoxin <0.1 EU/ug by LAL assay; A260/A280 ratio 1.80-1.95; >90% supercoiled by agarose gel; transfection efficiency >=80% of reference plasmid in HEK293T cells. |
| Key Features | Gravity-flow anion-exchange for transfection-grade plasmid; endotoxin <0.1 EU/ug; >90% supercoiled; scalable (25-100 mL culture); RNase A included; suitable for in vivo applications; no phenol or chloroform. |
| Purity | A260/A280 1.80-1.95; endotoxin <0.1 EU/ug DNA; <1% genomic DNA; <1% RNA; >90% supercoiled plasmid. |
| Concentration | Eluted plasmid at 0.5-2 ug/uL in TE or water (after resuspension). |
| Activity / Unit Definition | RNase A: specific activity >=70 Kunitz units/mg protein. |
| Molecular Weight | pUC19 reference plasmid: 2,686 bp, approximately 1.77x10^6 Da. |
| Source / Origin | Anion-exchange resin: synthetic hydrophilic polymer matrix with DEAE functional groups; all buffers: molecular biology grade chemicals; E. coli DH5alpha host strain recommended. |
| pH Range / Optimal pH | Buffer P1 pH 8.0; Buffer P2 pH 12.0-12.5; Buffer P3 pH 5.5; Buffer QC pH 7.0; Buffer QF pH 8.5. |
| Shipping Conditions | Ambient temperature; RNase A shipped at ambient (stable for 2 weeks at RT). |
| Expiration Date / Stability | 18 months for buffers and columns; once Buffer P1 is supplemented with RNase A, store at 2-8 C and use within 6 months. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. Plasmid DNA from this kit is suitable for research-grade transfection but not for GMP clinical-grade manufacturing. |
| Compatibility | Purified plasmid DNA compatible with: lipid-based transfection (Lipofectamine, FuGENE), calcium phosphate transfection, electroporation, microinjection, biolistic delivery, restriction digestion, Sanger sequencing, NGS library preparation, in vitro transcription, and PCR. Avoid repeated freeze-thaw cycles of purified plasmid. |
| Recommended Buffer System | P1: 50 mM Tris-HCl pH 8.0, 10 mM EDTA, 100 ug/mL RNase A; P2: 200 mM NaOH, 1% SDS; P3: 3.0 M potassium acetate pH 5.5; QC: 1.0 M NaCl, 50 mM MOPS pH 7.0, 15% isopropanol; QF: 1.25 M NaCl, 50 mM Tris-HCl pH 8.5, 15% isopropanol. |
| Application Notes / Precautions | For best results, use fresh overnight bacterial culture (12-16 hours growth). Do not exceed recommended culture volume or cell density to avoid column overloading. Gentle inversion (not vortexing) during alkaline lysis is critical to avoid shearing genomic DNA. Do not exceed 5 minutes for alkaline lysis step or supercoiled plasmid may denature. For low-copy plasmids, increase culture volume to 100 mL. If endotoxin removal is critical (e.g., primary cell transfection), use an additional endotoxin removal wash step. Store purified plasmid at -20 C in TE buffer for long-term stability. |
| Batch-to-Batch Consistency | Yield within +/-20% of reference pUC19 lot; endotoxin level <0.1 EU/ug for all lots; supercoiled percentage >90% for all lots. |
For research use only, not for clinical use.
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