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| Product Name | Yeast Genomic DNA Extraction Kit, Enzymatic Lysis, Spin Column |
| Catalog No. | DREK-0032 |
| Description | Spin column-based kit for rapid purification of high molecular weight genomic DNA from Saccharomyces cerevisiae (budding yeast), Schizosaccharomyces pombe (fission yeast), Pichia pastoris, and other fungal species. The protocol employs enzymatic cell wall digestion with recombinant lyticase (beta-1,3-glucanase) to generate spheroplasts, followed by proteinase K digestion and silica membrane column purification. The kit eliminates the need for glass bead beating, phenol-chloroform extraction, or CsCl gradient centrifugation. Purified genomic DNA is >50 kb in size with excellent purity and is suitable for PCR, qPCR, Southern blotting, whole genome sequencing, and long-read sequencing applications. |
| Intended Use | Purification of high molecular weight genomic DNA (>50 kb) from yeast and fungal cultures for PCR, qPCR genotyping, Southern blotting, restriction digestion, whole genome sequencing (Illumina, PacBio, Oxford Nanopore), and synthetic biology applications. |
| Principle / Technology | Enzymatic removal of yeast cell wall by recombinant lyticase producing osmotically fragile spheroplasts; spheroplast lysis by SDS and proteinase K; DNA binding to silica membrane in chaotropic salt buffer; wash steps remove polysaccharides, proteins, and other contaminants; elution in low-salt buffer releases purified high MW genomic DNA. |
| Detection Method | Harvest yeast cells (up to 2x10^8 cells) by centrifugation; wash cells; resuspend in sorbitol buffer with lyticase and beta-mercaptoethanol; incubate 30-60 min at 30 C (monitor spheroplast formation by OD600 in 1% SDS); centrifuge spheroplasts; lyse by adding lysis buffer and proteinase K, incubate 30 min at 56 C; add binding buffer and ethanol; load onto spin column; wash 2x; elute in 100-200 uL elution buffer or water. |
| Sample Type | Overnight yeast culture (up to 5 mL at OD600 ~5-10, or approximately 2x10^8 cells); S. cerevisiae, S. pombe, P. pastoris, Candida albicans, and filamentous fungi with appropriate pretreatment. |
| Performance Range / Specifications | Genomic DNA yield: 2-10 ug per 2x10^8 yeast cells; DNA fragment size: >50 kb (predominantly >100 kb); A260/A280 ratio 1.8-2.0; A260/A230 ratio >2.0; RNA-free by agarose gel electrophoresis. |
| Sensitivity / LOD | Detectable gDNA recovery from as few as 1x10^6 yeast cells; sufficient DNA for >100 PCR reactions at 10 ng per reaction. |
| Specificity | Lyticase specifically digests beta-1,3-glucan linkages in the yeast cell wall; silica membrane selectively binds double-stranded DNA; yeast mitochondrial DNA co-purifies with nuclear genomic DNA (represents <15% of total DNA). |
| Reaction Conditions / Protocol | Lyticase digestion: 30-60 min at 30 C; proteinase K digestion: 30 min at 56 C; column binding and wash: 10-15 min; total protocol time 1.5-2 hours. |
| Components / Formulation | Sorbitol Buffer (1.2 M sorbitol, pH 7.4), Lyticase (lyophilized, >=2,000 U), Proteinase K (20 mg/mL solution), Lysis Buffer, Binding Buffer, Wash Buffer 1, Wash Buffer 2 (concentrate), Elution Buffer (10 mM Tris-HCl, pH 8.5), Spin Columns with Collection Tubes, Protocol. |
| Storage Conditions | Lyticase and Proteinase K at -20 C; Sorbitol Buffer at 2-8 C; all other components at RT (15-25 C). |
| Shelf Life | 12 months from date of manufacture; Proteinase K stable at -20 C for 24 months. |
| Package Specifications | 20 preparations, 50 preparations. |
| Product Form | Liquid buffers; lyophilized lyticase; proteinase K solution; silica membrane spin columns. |
| Quality Control | Each lot tested for gDNA yield from S. cerevisiae W303 strain; A260/A280 ratio 1.8-2.0; DNA fragment size >50 kb by pulsed-field gel electrophoresis; PCR amplification of ACT1 gene from 1 ng gDNA template. |
| Key Features | Enzymatic cell wall lysis (no bead beating); high molecular weight DNA >50 kb; spin column format; phenol-free and chloroform-free; suitable for long-read sequencing; lyticase included. |
| Purity | A260/A280 1.8-2.0; A260/A230 >2.0; RNA-free; protein-free; polysaccharide-free. |
| Concentration | Typical yield 2-10 ug gDNA per preparation; post-elution concentration 20-100 ng/uL. |
| Activity / Unit Definition | Lyticase: >=2,000 units per vial (1 unit = amount producing a decrease in OD800 of 0.001 per minute at 25 C, pH 7.5); Proteinase K: >=30 mAnson units/mg. |
| Molecular Weight | S. cerevisiae genome: approximately 12.1 Mb (haploid), 1.2x10^7 bp. |
| Source / Origin | Lyticase: recombinant from Arthrobacter luteus expressed in E. coli; Proteinase K: from Tritirachium album expressed in Pichia pastoris; silica membrane: synthetic. |
| pH Range / Optimal pH | Sorbitol buffer pH 7.4; lysis buffer pH 8.0-8.5 (optimal for proteinase K); binding buffer pH 5.0-6.0; elution buffer pH 8.5. |
| Shipping Conditions | Ambient temperature for most components; lyticase and proteinase K on blue ice (-20 C). |
| Expiration Date / Stability | 12 months at recommended conditions; proteinase K solution stable at 2-8 C for 12 months after thawing; lyticase stable at -20 C desiccated; avoid repeated freeze-thaw cycles. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. |
| Compatibility | Purified gDNA compatible with PCR, qPCR, restriction digestion, Southern blotting, whole genome sequencing (Illumina, PacBio, Oxford Nanopore), and synthetic biology cloning applications. For Qubit or PicoGreen quantification, use dsDNA-specific fluorescent dyes as A260 measurement may overestimate concentration due to RNA co-purification (though RNA levels are low). Elution buffer is EDTA-free for magnesium-dependent enzymatic reactions. |
| Recommended Buffer System | Sorbitol Buffer: 1.2 M sorbitol, 50 mM potassium phosphate pH 7.4, 0.5 mM beta-mercaptoethanol; Lysis: 50 mM Tris-HCl pH 8.0, 50 mM EDTA, 1% SDS; Binding: guanidine HCl, isopropanol, pH 5-6. |
| Application Notes / Precautions | Monitor spheroplast formation by mixing 10 uL cell suspension with 990 uL 1% SDS and measuring OD600 — spheroplast formation is indicated by >80% reduction in OD600 compared to untreated cells. For filamentous fungi, additional mechanical disruption may be needed. Young cultures (early log phase) yield spheroplasts more easily than stationary phase cultures. Include beta-mercaptoethanol in sorbitol buffer for reducing disulfide bonds in the cell wall. After elution, incubate elution buffer on column for 2-5 minutes at RT for maximum yield. Store purified gDNA at -20 C (short term) or -80 C (long term); avoid repeated freeze-thaw cycles. |
| Batch-to-Batch Consistency | gDNA yield within +/-20% of reference lot; A260/A280 ratio within 1.8-2.0; PCR amplification Cq within +/-0.5 cycles. |
For research use only, not for clinical use.
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