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| Product Name | Large-Scale Plasmid DNA Extraction Kit (Maxi/Mega Prep) |
| Catalog No. | DREK-0022 |
| Description | An anion-exchange resin-based plasmid DNA purification kit for the large-scale isolation of transfection-grade plasmid DNA from high-volume bacterial cultures. The gravity-flow column format accommodates high lysate loading volumes, and the anion-exchange chemistry provides selective plasmid DNA binding while removing RNA, genomic DNA, proteins, and endotoxins. The isopropanol precipitation recovery step concentrates the purified plasmid, making it suitable for demanding applications including stable cell line generation, in vivo gene delivery studies, and large-scale transfection experiments. |
| Intended Use | Large-scale purification of transfection-grade plasmid DNA from 100-500 mL (Maxi) or 500-2500 mL (Mega) E. coli cultures for mammalian cell transfection, in vivo gene delivery, microinjection, gene therapy vector preparation, and other applications requiring milligram quantities of high-purity, low-endotoxin plasmid DNA. |
| Principle / Technology | Bacterial cells are harvested and subjected to modified alkaline lysis. After neutralization, the cleared lysate is loaded by gravity flow onto a pre-equilibrated anion-exchange resin column. Plasmid DNA binds to the positively charged resin under moderate salt conditions while RNA, proteins, and metabolites flow through. An endotoxin removal wash step is incorporated before eluting plasmid DNA under high-salt conditions. Isopropanol precipitation recovers and concentrates the plasmid, which is washed with ethanol and resuspended in TE buffer or water. |
| Detection Method | UV spectrophotometry (A260/A280, A260/A230); agarose gel electrophoresis for supercoiled/nicked form analysis; restriction enzyme digestion; fluorometric quantitation; LAL endotoxin assay; transfection efficiency evaluation in mammalian cell lines; Sanger sequencing of representative regions |
| Sample Type | E. coli cultures grown in LB, TB, or 2xYT medium (100-500 mL for Maxi, 500-2500 mL for Mega); suitable for high-copy (pUC origin) and low-copy plasmids in DH5α, TOP10, XL1-Blue, Stbl3, and other standard cloning strains |
| Performance Range / Specifications | Maxi: 100-500 mL culture; yield: 500-1500 µg (high-copy), 100-300 µg (low-copy); Mega: 500-2500 mL culture; yield: 2.5-10 mg (high-copy), 500 µg-2 mg (low-copy); A260/A280: 1.80-1.95; A260/A230: >1.8; >85% supercoiled; endotoxin <0.1 EU/µg; transfection efficiency ≥70% in HEK293 cells |
| Sensitivity / LOD | Plasmid recovery from cultures with OD600 as low as 1.5; detectable plasmid from low-copy constructs; endotoxin levels consistently below LAL assay sensitivity after standard protocol |
| Specificity | Purified plasmid DNA free of genomic DNA, RNA, and protein; endotoxin removal step achieves <0.1 EU/µg; no detectable RNase or DNase activity in final product; restriction digestion produces expected fragment pattern; sequencing confirms insert integrity |
| Reaction Conditions / Protocol | Maxi: harvest 100-500 mL culture at 6,000g for 15 minutes at 4°C; resuspend in 10 mL resuspension buffer; add 10 mL lysis buffer; mix gently; incubate 5 minutes; add 10 mL chilled neutralization buffer; mix; incubate on ice 15 minutes; filter lysate through supplied filtration unit; load onto pre-equilibrated column; allow to flow through by gravity; wash with 2 × 30 mL wash buffer; wash with 2 × 15 mL endotoxin removal buffer; elute with 15 mL elution buffer; precipitate with 0.7 volumes isopropanol; centrifuge at 15,000g for 30 minutes at 4°C; wash with 70% ethanol; air-dry; resuspend in 0.5-1 mL TE; total time: 4-5 hours. Mega: scale proportionally with larger column format and buffer volumes. |
| 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), equilibration buffer, wash buffer, endotoxin removal buffer, elution buffer (high-salt), isopropanol, 70% ethanol (user-supplied), anion-exchange resin columns (Maxi and Mega sizes), lysate filtration units, endotoxin-free collection tubes |
| Storage Conditions | Resuspension buffer with RNase A at 2-8°C; lysis and neutralization buffers at room temperature; column equilibration, wash, and elution buffers at room temperature; protected columns and buffers from light; isopropanol at room temperature |
| Shelf Life | 24 months for buffers and columns; resuspension buffer with RNase A stable 6 months at 2-8°C; buffers stable 12 months after opening at room temperature |
| Package Specifications | 5 Maxi preparations and 25 Maxi preparations; 5 Mega preparations; all consumables included; isopropanol and ethanol not supplied |
| Product Form | Pre-packed anion-exchange resin columns; liquid buffers in bulk bottles; lysate filtration syringes; RNase A pre-added to resuspension buffer; endotoxin removal buffer |
| Quality Control | Each lot validated with pCMV-GFP control plasmid: Maxi yield ≥500 µg; A260/A280: 1.80-1.95; >85% supercoiled; endotoxin <0.1 EU/µg; transfection efficiency ≥70% in HEK293; no gDNA by PCR; restriction digestion confirms expected pattern; Sanger sequencing coverage >98% |
| Key Features | Anion-exchange chemistry for high-purity transfection-grade DNA; patent-protected endotoxin removal technology; gravity-flow format requires no pumps or FPLC; yields suitable for milligram-scale applications; consistent performance with high-copy and low-copy plasmids; endotoxin levels appropriate for in vivo and clinical research applications |
| Purity | A260/A280: 1.80-1.95; A260/A230: >1.8; protein <0.2%; RNA <1%; genomic DNA undetectable; endotoxin <0.1 EU/µg |
| Concentration | Resuspension 0.5-1 mL (Maxi) or 2-5 mL (Mega); typical concentration 0.5-2 µg/µL for final resuspension |
| Activity / Unit Definition | Not applicable for plasmid purification kits |
| Molecular Weight | Not applicable; plasmid molecular weight varies with construct size typically 2-20 kb |
| Source / Origin | Anion-exchange resin is a proprietary cross-linked polyacrylate matrix with quaternary ammonium functional groups; RNase A from bovine pancreas; all buffers are molecular biology grade; columns are endotoxin-free certified |
| 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; equilibration/wash buffers pH 7.0-7.5; elution buffer pH 8.0-8.5 |
| Shipping Conditions | Ambient temperature for all components; columns shipped in protective packaging; no dangerous goods classification; standard freight acceptable |
| Expiration Date / Stability | 24 months unopened; column resin stability verified by binding capacity testing at 0, 12, and 24 months; resuspension buffer with RNase A: 6 months at 2-8°C after first use; endotoxin removal buffer verified effective for 24 months sealed |
| Regulatory / Compliance | For research use only; suitable for preclinical and translational research applications; endotoxin specification <0.1 EU/µg appropriate for sensitive cell types; not manufactured under GMP for clinical applications |
| Compatibility | Purified plasmid suitable for mammalian cell transfection (calcium phosphate, lipid-based, electroporation), in vivo gene delivery, microinjection, in vitro transcription, and restriction analysis; anion-exchange column can be regenerated and reused (up to 5 uses) with appropriate cleaning protocol |
| Recommended Buffer System | Tris-EDTA resuspension; NaOH-SDS alkaline lysis; potassium acetate neutralization; anion-exchange (quaternary ammonium) binding at moderate salt; high-salt elution; isopropanol precipitation recovery |
| Application Notes / Precautions | Pre-chill neutralization buffer before use for optimal genomic DNA precipitation; do not vortex after lysis step; use endotoxin-free tubes for final resuspension; for largest plasmids (>15 kb), gentle inversion during lysis is critical; monitor column flow rate—do not apply pressure; for low-copy plasmids, increase culture volume within column capacity limits; repeat isopropanol precipitation if higher concentration is needed |
| Batch-to-Batch Consistency | Plasmid yield CV <15% between lots; column binding capacity >2.5 mg plasmid DNA per Maxi column; endotoxin removal verified <0.1 EU/µg per lot; supercoiled percentage >85% per batch; transfection efficiency validation per lot; resin functional group density controlled per manufacturing batch |
For research use only, not for clinical use.
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