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| Product Name | Magnetic Bead-Based Genomic DNA Extraction Kit (Bacteria) |
| Catalog No. | DREK-0005 |
| Description | A magnetic bead-based genomic DNA purification kit optimized for the extraction of high-quality genomic DNA from both Gram-positive and Gram-negative bacterial species. The kit includes a dedicated lysozyme-based pretreatment step with optimized enzymatic digestion conditions that efficiently disrupt the peptidoglycan cell wall of Gram-positive organisms. For Gram-negative bacteria, a simplified boiling-lysis protocol can be substituted. The magnetic silica bead technology enables rapid, centrifugation-free purification suitable for high-throughput applications. |
| Intended Use | Isolation of bacterial genomic DNA from pure cultures of Gram-positive and Gram-negative bacteria, including clinically relevant species with difficult-to-lyse cell walls, for 16S rRNA gene sequencing, whole-genome sequencing, antimicrobial resistance gene detection, and molecular epidemiology studies. |
| Principle / Technology | Bacterial cells are harvested by centrifugation and resuspended in an enzymatic lysis buffer containing lysozyme, mutanolysin, and RNase A. After incubation, SDS and proteinase K are added for complete protein digestion and DNA release. Following clarification, binding buffer is added to promote DNA binding to silica-coated magnetic beads. After washing to remove cell debris, proteins, and heme from blood-derived isolates, purified DNA is eluted in Tris-EDTA buffer. |
| Detection Method | UV spectrophotometry; agarose gel electrophoresis; fluorometric quantitation; 16S rRNA gene PCR amplification and sequencing for species-level confirmation; whole-genome sequencing quality metrics (coverage, N50) for representative isolates |
| Sample Type | Pure bacterial cultures from solid agar plates or liquid broth; Gram-negative species (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella enterica) and Gram-positive species (Staphylococcus aureus, Streptococcus pneumoniae, Bacillus subtilis, Enterococcus faecalis, Mycobacterium smegmatis); bacterial pellets from 1-5 mL overnight culture |
| Performance Range / Specifications | Input: bacterial pellet from 1-5 mL overnight culture (OD600 0.6-3.0); yield: 5-15 µg from E. coli 3 mL culture, 3-8 µg from S. aureus 3 mL culture, 1-3 µg from M. smegmatis 3 mL culture; A260/A280: 1.80-1.95; A260/A230: >1.8; fragment size: 30-50 kb |
| Sensitivity / LOD | Effective DNA recovery from as few as 10^5 bacterial cells; consistently detects single-copy genomic targets by qPCR; recovers DNA from stationary-phase and biofilm-state bacteria |
| Specificity | Bacterial genomic DNA free of co-purified RNA (RNase A treatment included); minimal plasmid DNA carryover for high-copy plasmid-bearing strains; DNA free of PCR inhibitors including bile salts and blood components from clinical isolates; genus- and species-level identification confirmed by 16S rRNA sequencing |
| Reaction Conditions / Protocol | Harvest 1-5 mL bacterial culture by centrifugation at 5,000g for 5 minutes; resuspend pellet in enzymatic lysis buffer (lysozyme, mutanolysin, RNase A in Tris-EDTA); incubate at 37°C for 30-60 minutes (extend to 2 hours for Gram-positive species); add SDS and proteinase K; incubate at 56°C for 30 minutes; add binding buffer and isopropanol; add magnetic bead suspension; incubate 10 minutes at room temperature; separate beads magnetically; wash with wash buffer 1 and twice with wash buffer 2; elute in 50-100 µL Tris-EDTA at 56°C; total time: 2-3.5 hours depending on species |
| Components / Formulation | Enzymatic lysis buffer (lysozyme 20 mg/mL, mutanolysin 250 U/mL, RNase A 100 µg/mL in 20 mM Tris-HCl pH 8.0, 2 mM EDTA), SDS solution (10%), proteinase K (20 mg/mL), binding buffer (guanidine hydrochloride, Tris-HCl, isopropanol), magnetic silica bead suspension (50 mg/mL), wash buffer 1 concentrate, wash buffer 2 concentrate, elution buffer (10 mM Tris-HCl pH 8.5, 0.5 mM EDTA), magnetic separation rack |
| Storage Conditions | Lysozyme, mutanolysin, RNase A lyophilized stored at -20°C; reconstituted enzymes at 2-8°C for up to 4 weeks; proteinase K at 2-8°C; buffers at room temperature; magnetic beads at 2-8°C |
| Shelf Life | 24 months for buffers; lyophilized enzymes 24 months at -20°C; reconstituted enzymes 4 weeks at 2-8°C; magnetic bead suspension 18 months at 2-8°C |
| Package Specifications | 50 preparations and 200 preparations; lysozyme, mutanolysin, RNase A provided as lyophilized powders; all buffers and magnetic beads included; magnetic rack sold separately |
| Product Form | Liquid buffers; silica-coated magnetic bead suspension; lyophilized enzymes (reconstitute before use); proteinase K solution |
| Quality Control | Each lot tested with E. coli ATCC 25922 and S. aureus ATCC 25923: yield ≥5 µg and ≥3 µg respectively; A260/A280: 1.80-1.95; 16S rRNA PCR amplification confirmed; no PCR inhibition in multiplex assay; Gram-positive lysis efficiency >90% confirmed by qPCR of released DNA vs heat-treated control |
| Key Features | Dual-enzyme lysis system for broad Gram-positive coverage; RNase A co-treatment eliminates separate RNA removal step; magnetic bead format allows high-throughput processing in 96-well format; inhibitor-free DNA suitable for NGS without additional cleanup; effective with clinical isolates including mucoid and capsule-producing strains |
| Purity | A260/A280: 1.80-1.95; A260/A230: >1.8; protein contamination <0.5% by Bradford assay |
| Concentration | DNA concentration 50-200 ng/µL in 50-100 µL elution; yield dependent on bacterial species and growth phase |
| Activity / Unit Definition | Not applicable for extraction kits |
| Molecular Weight | Not applicable for genomic DNA from bacterial sources |
| Source / Origin | Silica-coated magnetic beads from synthetic sol-gel process; lysozyme from hen egg white; mutanolysin from Streptomyces globisporus; proteinase K recombinantly expressed in Pichia pastoris; RNase A from bovine pancreas |
| pH Range / Optimal pH | Enzymatic lysis buffer pH 7.8-8.2; binding buffer pH 5.5-6.5; wash buffers pH 6.5-7.5; elution buffer pH 8.3-8.7 |
| Shipping Conditions | Ambient temperature for buffers and beads; enzymes shipped on dry ice; proteinase K shipped with cold packs |
| Expiration Date / Stability | 24 months for buffers and dry enzymes; real-time and accelerated stability data available; reconstituted enzyme activity maintains >80% after 4 weeks at 2-8°C |
| Regulatory / Compliance | For research use only; suitable for molecular epidemiology and microbial genomics research; not for clinical diagnostic use |
| Compatibility | DNA suitable for 16S rRNA gene sequencing, whole-genome sequencing (Illumina, PacBio, Oxford Nanopore), MLST, PCR-based detection, and qPCR quantification; compatible with KingFisher and other automated magnetic bead handlers |
| Recommended Buffer System | Tris-EDTA enzymatic lysis buffer with SDS; guanidine hydrochloride binding chemistry; Tris-EDTA elution |
| Application Notes / Precautions | For mycobacterial species, extend lysozyme incubation to 3-4 hours and include a 10-minute 80°C heat inactivation step; for mucoid Pseudomonas, increase initial culture centrifugation speed; avoid vortexing after DNA elution to maintain high molecular weight; for low-biomass samples, reduce elution volume to 20-30 µL; complete removal of wash buffer 2 before elution is critical for downstream enzymatic reactions |
| Batch-to-Batch Consistency | Between-lot yield CV <15% for E. coli and S. aureus reference strains; A260/A280 <0.05 variation between lots; enzymatic lysis activity verified per batch for lysozyme (>20,000 U/mg) and mutanolysin (>2,000 U/mg); magnetic bead binding capacity verified at ≥10 µg DNA per preparation |
For research use only, not for clinical use.
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