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| Product Name | Column-Based Bacterial Genomic DNA Extraction Kit |
| Catalog No. | DREK-0017 |
| Description | A silica membrane spin column kit designed for the isolation of genomic DNA from Gram-negative and Gram-positive bacterial cultures. The kit includes lysozyme for enzymatic digestion of the peptidoglycan cell wall, along with proteinase K and SDS for protein removal. The column-based format provides a fast, reproducible workflow suitable for daily bacterial genomics work including 16S rRNA gene analysis and whole-genome sequencing library preparation. |
| Intended Use | Extraction of genomic DNA from bacterial pure cultures for molecular identification by 16S rRNA gene sequencing, whole-genome sequencing, antimicrobial resistance gene detection, plasmid profiling, and molecular epidemiological typing. |
| Principle / Technology | Bacteria are pelleted and subjected to enzymatic lysis with lysozyme in a Tris-EDTA buffer to degrade the peptidoglycan layer. SDS and proteinase K are added for protein digestion and complete cell lysis. After incubation, a guanidine hydrochloride-based binding buffer and ethanol are added, and the lysate is passed through a silica membrane column. DNA binds to the membrane, and after wash steps, purified DNA is eluted in a low-salt buffer. |
| Detection Method | UV spectrophotometry for purity; agarose gel electrophoresis for molecular weight; fluorometric quantitation; 16S rRNA gene PCR for identity confirmation; whole-genome sequencing quality metrics; restriction enzyme digestion for DNA quality assessment |
| Sample Type | Pure bacterial cultures from agar plates or liquid broth; Gram-negative (E. coli, Pseudomonas, Salmonella, Klebsiella, Acinetobacter), Gram-positive (Staphylococcus, Streptococcus, Bacillus, Enterococcus, Listeria), and mycobacterial species (with modified protocol); cultures grown in standard bacteriological media |
| Performance Range / Specifications | Input: pellet from 1-3 mL overnight culture; yield: 5-15 µg from E. coli (Gram-negative), 3-10 µg from S. aureus (Gram-positive); A260/A280: 1.80-1.95; column binding capacity: 25 µg; fragment size: 30-50 kb; RNA-free with RNase A treatment included |
| Sensitivity / LOD | Detectable DNA from as few as 10⁵ bacterial cells; consistent recovery from slow-growing organisms; adequate DNA yield for whole-genome sequencing from a single 3 mL culture of most enteric bacteria |
| Specificity | Pure bacterial genomic DNA; no eukaryotic DNA contamination; no fungal DNA (unless processing fungal samples); RNase A treatment ensures RNA-free preparation; no PCR inhibitors; restriction digest shows expected patterns without anomalous bands |
| Reaction Conditions / Protocol | Harvest bacteria from 1-3 mL overnight culture at 5,000g for 5 minutes; resuspend in 180 µL enzymatic lysis buffer (lysozyme in Tris-EDTA); incubate at 37°C for 30 minutes (Gram-negative) or 60 minutes (Gram-positive); add 20 µL proteinase K and 200 µL lysis/binding buffer; vortex; incubate at 56°C for 30 minutes; add 200 µL ethanol; vortex; transfer to spin column; centrifuge at 8,000g for 1 minute; wash with 500 µL wash buffer; centrifuge; wash with 500 µL wash buffer; centrifuge 3 minutes; elute with 50-100 µL elution buffer; incubate at room temperature 5 minutes; centrifuge; total time: 1.5-2.5 hours |
| Components / Formulation | Lysozyme (lyophilized, ≥20,000 U/mg), enzymatic lysis buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 1.2% Triton X-100), proteinase K solution (20 mg/mL), lysis/binding buffer (guanidine hydrochloride, Tris-HCl, EDTA, SDS), 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; RNase A (optional, not supplied) |
| Storage Conditions | Lysozyme at -20°C lyophilized, 2-8°C up to 4 weeks after reconstitution; proteinase K at 2-8°C; all buffers at room temperature; columns at room temperature in sealed pouch |
| Shelf Life | 24 months for buffers and columns; lysozyme 24 months lyophilized at -20°C; proteinase K 12 months at 2-8°C after opening; reconstituted lysozyme 4 weeks at 2-8°C |
| Package Specifications | 50 preparations and 250 preparations; includes lysozyme, proteinase K, and all buffers; spin columns with collection tubes included |
| Product Form | Silica membrane spin columns; liquid buffers; lyophilized lysozyme requiring reconstitution; proteinase K solution |
| Quality Control | Each lot validated with E. coli ATCC 25922 and S. aureus ATCC 25923: yield ≥5 µg and ≥3 µg; A260/A280: 1.80-1.95; 16S rRNA PCR positive; EcoRI digestion complete; DNase/RNase free; no PCR inhibitors; lot traceability for enzymes including activity certification |
| Key Features | Broad-spectrum bacterial lysis system; lysozyme included for Gram-positive organisms; optimized binding buffer for bacterial DNA; high-quality DNA for NGS without additional purification; room temperature buffer storage; consistent inter-sample performance |
| Purity | A260/A280: 1.80-1.95; A260/A230: >1.8; RNA contamination <2% after RNase treatment; protein <0.3% |
| Concentration | Elution volume 50-100 µL; DNA concentration 50-200 ng/µL for Gram-negative; 30-100 ng/µL for Gram-positive species |
| Activity / Unit Definition | Not applicable for DNA extraction kits |
| Molecular Weight | Not applicable for bacterial genomic DNA of heterogeneous size |
| Source / Origin | Silica membrane from borosilicate glass microfiber; lysozyme from hen egg white (salt-free, chromatographically purified); proteinase K from recombinant expression; all buffers chemically synthesized, nuclease-free |
| pH Range / Optimal pH | Enzymatic lysis buffer pH 7.8-8.2; lysis/binding buffer pH 5.8-6.5; wash buffer pH 7.0-7.5; elution buffer pH 8.3-8.7 |
| Shipping Conditions | Ambient temperature for buffers and columns; lysozyme and proteinase K shipped with cold packs or on dry ice |
| Expiration Date / Stability | 24 months for buffers and columns; lysozyme maintains >90% activity at -20°C for 24 months; proteinase K stable 12 months at 2-8°C; accelerated stability at 37°C confirms shelf-life claims; buffer pH stable throughout shelf life |
| Regulatory / Compliance | For research use only; suitable for bacterial genomics, microbiome research, and antimicrobial resistance surveillance research; not for clinical diagnostic applications; appropriate biosafety containment for pathogenic organisms is the user's responsibility |
| Compatibility | Eluted DNA suitable for 16S rRNA gene amplification and sequencing, whole-genome sequencing (Illumina, PacBio, Oxford Nanopore), MLST, PCR-based detection, qPCR, and Southern blotting; compatible with vacuum manifold for 96-well plate processing |
| Recommended Buffer System | Tris-EDTA-Triton enzymatic lysis; guanidine hydrochloride-SDS binding and digestion; ethanol-based wash; Tris-EDTA elution |
| Application Notes / Precautions | For mucoid or encapsulated bacteria, increase lysozyme incubation time and concentration; for mycobacteria, pretreatment with glycine and lysozyme at 37°C for 12-16 hours is recommended; ensure complete resuspension of bacterial pellet before adding enzymes; Gram-positive lysis efficiency can be monitored by increased viscosity after SDS addition; avoid excessive drying of column membrane; for species producing high levels of DNase, process samples promptly after harvest |
| Batch-to-Batch Consistency | DNA yield CV <15% for E. coli and S. aureus between lots; A260/A280 variation <0.04; lysozyme specific activity verified per batch (>20,000 U/mg); column binding capacity verified >25 µg per lot; buffer pH and conductivity controlled per batch |
For research use only, not for clinical use.
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