pLenti-CBLC-sgRNA

Cat. No.: P744452

Specifications

Product Name

pLenti-CBLC-sgRNA

Product Line

생물 제품

보관 및 배송

보관 조건

80°C에서 보관

배송

Dry ice packs + Cold packs

안정성 및 스토리지

20℃에서 보관하면 6개월 이상 유효하며, -80℃에서 보관하면 1년 이상 유효합니다.

Application Protocols

• Only product data may be listed here. No tested applications or recommended conditions are specified for this item in the catalog entry.
• Refer to the CoA/Spec Sheet for any lot-specific guidance. General procedures for cloning, transfection, or lentiviral packaging should follow institutional SOPs and reagent manufacturers’ instructions.

Biological Roles

• Item-specific biological role: Not specified for this item; refer to CoA/Spec Sheet.
• General context (literature/general)
  • sgRNA (single guide RNA) directs CRISPR-associated nucleases (e.g., Cas9) to specific genomic loci via base pairing with the target DNA sequence adjacent to an appropriate PAM. This enables site‑specific double‑strand breaks (DSBs) or, with catalytically dead Cas9 fusions, transcriptional modulation without cleavage.
  • Lentiviral vectors deliver sgRNA expression cassettes efficiently to a wide range of mammalian cells, including non‑dividing populations, supporting stable integration and long‑term expression when used with appropriate packaging systems.
  • If “CBLC” in the product name refers to the Cbl proto‑oncogene C (CBLC) locus, sgRNAs would typically be designed to modulate or disrupt that gene; the exact target sequence and species for this item are not provided here and must be confirmed from the sequence map/CoA.

Buffer Applications

• Not typically applicable. This product is not a buffering agent.
• Practical handling buffers (literature/general)
  • Common storage/dilution matrices for plasmid DNA include nuclease‑free water or TE buffer (10 mM Tris-Cl, pH 7.5–8.0, 0.1–1 mM EDTA). Exact buffer for this item is not specified.
  • For enzymatic reactions (e.g., restriction digest, ligation), use the enzyme supplier’s recommended buffers to maintain activity and fidelity.

Green Alternatives

• Not directly applicable to a plasmid DNA reagent. However, sustainability considerations can be applied to workflows using this product (literature/general):
  • Use enzymatic DNA cleanup (spin columns, magnetic beads) instead of organic solvent–intensive phenol–chloroform extractions where possible.
  • Select transfection reagents with lower cytotoxicity and reduced hazardous waste generation; minimize use of halogenated solvents in any ancillary steps.
  • Consolidate shipments and store long‑term aliquots at −80 °C to reduce repeated cold-chain shipments and waste.
• Comparison (literature/general)
  • Phenol–chloroform vs. column/bead cleanup: columns/beads eliminate toxic organic phases, reduce hazardous waste, and streamline processing, at modest consumable cost.
  • Calcium phosphate transfection vs. modern lipids/polymers: newer reagents often yield higher efficiency at neutral pH and produce less particulate waste; choose formulations compatible with your cell line to reduce repeats.

Pharmaceutical Uses

• Not applicable for clinical/therapeutic use. For research use only, as stated by the manufacturer.
• General formulation context (literature/general)
  • Plasmid DNA and lentiviral vectors are widely used in research and bioprocess development for gene function studies, cell line engineering, and assay development. Any use in GMP or clinical settings requires dedicated, validated manufacturing and quality systems distinct from standard research-grade materials.
  • No pharmacopeial monograph or excipient status is implied for this item.

Physical Properties

• Item-specific specifications
  • Concentration, A260/280, buffer composition: Not specified for this item; refer to CoA/Spec Sheet.
  • Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• General properties for plasmid DNA (literature/general)
  • State: Aqueous solution or lyophilized solid of double‑stranded circular DNA, typically colorless in solution.
  • Solubility: Readily soluble in aqueous buffers (e.g., TE, Tris, nuclease‑free water). Insoluble in most nonpolar organic solvents.
  • Stability: Stable to multiple freeze–thaw cycles when aliquoted and stored at ≤ −20 °C; best practice is storage at −80 °C to minimize nuclease activity and depurination over time.
  • UV characteristics: DNA exhibits λmax ~260 nm with typical A260/280 ~1.8–2.0 for pure DNA (literature values; not a specification for this item).
  • Diffusion/viscosity: High molecular weight; viscosity increases with concentration and supercoiling state (literature/general).
• Handling implications (literature/general)
  • Avoid repeated freeze–thaw without aliquoting; use nuclease‑free plasticware and solutions to prevent degradation.
  • DNase contamination and residual salts/solvents from preparation can impact downstream performance; verify by gel or capillary electrophoresis and spectrophotometry as needed.

Quality and Grades

• Item-specific grade/purity: Not specified for this item; refer to CoA/Spec Sheet.
• General quality considerations for plasmid DNA (literature/general)
  • Endotoxin level: For transfection or in vivo research, low endotoxin preparations are preferred (e.g., <0.1 EU/µg DNA). For routine cloning, standard molecular biology grade may suffice.
  • Supercoiling/topology: High percentage of supercoiled (ccc) form supports efficient transfection and packaging. Nicked or linear forms may reduce performance.
  • Sequence verification: Sanger or NGS confirmation of the sgRNA protospacer and vector junctions is standard to ensure target fidelity and absence of unintended mutations.
  • Residual contaminants: Low residual salts, phenol, ethanol, and RNase/DNase. A260/230 ratio near ~2.0 is typical for clean DNA (literature values; not specifications for this item).
• Stabilizers and formulation
  • DNA is commonly supplied in nuclease‑free water or TE buffer (10 mM Tris, 0.1–1 mM EDTA) to enhance stability; exact buffer for this item is not specified. EDTA can chelate Mg2+ and inhibit nucleases but may be incompatible with some enzymatic reactions at high concentration.

Reaction and Applications

• Item-specific applications: Not specified for this item; refer to CoA/Spec Sheet and catalog datasheet.
• General applications for pLenti sgRNA vectors (literature/general)
  • CRISPR gene editing: Expression of sgRNA to guide a co-expressed or separately supplied Cas nuclease (e.g., SpCas9) for targeted genome editing in mammalian cells.
  • Gene modulation: When paired with dCas9 effectors, sgRNA vectors can support CRISPRi (transcriptional repression) or CRISPRa (activation) screens.
  • Lentiviral delivery workflows: The plasmid can be used as a transfer vector in a three- or four‑plasmid packaging system to generate replication‑incompetent lentiviral particles for efficient delivery to dividing and non‑dividing cells (user‑performed; institutional approvals required).
  • Library/screening: Derivatives enable pooled sgRNA libraries for loss‑of‑function or perturb-seq studies.
• Practical tips (literature/general)
  • Verify the sgRNA protospacer sequence and PAM compatibility with the selected nuclease and target species genome before use.
  • Use high-quality, endotoxin‑reduced DNA for transfection or viral packaging. Confirm insert by restriction digest and Sanger sequencing.
  • Include appropriate non-targeting and positive‑control guides in experiments to benchmark editing efficiency.

Reaction Conditions

• Item-specific conditions: Not specified for this item; refer to CoA/Spec Sheet or the lab’s validated SOPs.
• General literature guidance for common workflows (not product specifications)
  • Bacterial propagation: Typical E. coli cloning strains grown at 30–37 °C in appropriate antibiotic selection; plasmid minipreps/midipreps per kit instructions.
  • Transfection (plasmid delivery): Mammalian cells at 37 °C, 5% CO2; DNA:reagent ratios per transfection reagent guidance; medium change 4–24 h post‑transfection as appropriate.
  • Lentiviral packaging (if applicable): Third‑generation packaging in HEK293T cells with helper plasmids; harvest supernatant 48–72 h post‑transfection; optional concentration by PEG or ultracentrifugation. Observe BSL‑2 practices and institutional approvals.
  • CRISPR editing window: Assess indels by 48–96 h post‑delivery; for stable lines, select and expand clones prior to genotyping. Editing efficiency depends on guide design, nuclease, chromatin context, and delivery method.
  • Storage/handling: Maintain DNA on ice during setup; avoid repeated freeze–thaw by aliquoting. Use nuclease‑free consumables.

Safety and Handling

• Item-specific hazard data
  • GHS classification, signal word, H-statements, pictograms: Not specified for this item; consult the SDS.
• General safety for plasmid DNA and lentiviral backbones (literature/general)
  • Plasmid DNA itself is generally considered nonhazardous; however, constructs intended for lentiviral packaging should be handled in accordance with institutional biosafety procedures. Packaging into replication‑incompetent lentiviral particles (if performed by the user) typically requires BSL‑2 containment and appropriate engineering controls.
  • Avoid ingestion, inhalation of aerosols, or skin/eye contact with solutions. Use standard microbiological practices.
• PPE and engineering controls (literature/general)
  • Recommended PPE: lab coat, nitrile gloves, and eye protection. Work in a certified biological safety cabinet when handling materials that may generate aerosols or when conducting viral packaging steps.
• Incompatibilities and precautions (literature/general)
  • Avoid nuclease contamination; keep solutions on ice during handling; use nuclease‑free consumables. Do not autoclave plasmid DNA solutions intended for transformation or transfection.
• First aid (overview; defer to SDS)
  • Skin/eye contact: rinse with water. If irritation persists, seek medical attention. Ingestion/inhalation: move to fresh air, rinse mouth, and seek medical advice. Always consult the SDS for authoritative guidance.

Solvent Selection

• Not typically applicable. This product is a nucleic acid reagent, not used as a solvent.
• Practical notes (literature/general)
  • Compatible media: nuclease‑free water or low‑salt Tris buffers for dilution. Avoid organic solvents that denature DNA (e.g., phenol, chloroform, alcohols) except where used for purification and fully removed prior to biological applications.
  • If concentrating or desalting, use spin columns or ethanol/isopropanol precipitation followed by resuspension in the desired aqueous buffer.

Storage and Reconstitution

• Item-specific instructions
  • Storage conditions: Store at −80 °C (as provided in Product Data).
  • Shipped in: Dry ice packs + Cold packs (as provided in Product Data) to maintain frozen/cold chain integrity.
  • Appearance, buffer, concentration, and reconstitution (if lyophilized): Not specified for this item; refer to CoA/Spec Sheet.
• General guidance (literature/general)
  • Upon receipt, verify package integrity and immediately transfer to −80 °C. If frequent access is expected, prepare single-use aliquots to minimize freeze–thaw cycles.
  • If lyophilized, reconstitute with nuclease‑free water or TE buffer to the desired concentration; mix gently by pipetting. Allow complete dissolution on ice. Avoid vigorous vortexing that can shear DNA.
  • Record freeze–thaw cycles and avoid repeated thawing. Store working aliquots at −20 °C (short term) and master stock at −80 °C (long term). Use nuclease‑free tubes and tips throughout.

Structure and Identity

• Item-specific identifiers
  • Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
  • Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.
  • SMILES/InChI/InChIKey/CAS/CID: Not specified for this item; refer to CoA/Spec Sheet.
• General description (literature/typical for such products)
  • pLenti-CBLC-sgRNA denotes a plasmid DNA construct in a lentiviral backbone configured to express a single guide RNA (sgRNA). The “pLenti” designation typically indicates a third-generation lentiviral transfer vector backbone compatible with packaging into VSV-G–pseudotyped lentiviral particles (literature/general).
  • sgRNA cassettes are commonly driven by U6/H1 RNA polymerase III promoters and contain a guide sequence plus scaffold tracr sequence recognized by Cas9 or other CRISPR nucleases (literature/general).
  • Structural features (conceptual, literature): covalently closed circular, double-stranded DNA; modular elements usually include promoter(s), selectable marker(s), an origin of replication, and an sgRNA scaffold region. Exact map, guide sequence, and antibiotic markers for this item are not specified; consult the sequence map/CoA.
• 2D/functional view (literature/general)
  • A circular plasmid map with discrete blocks: bacterial ori, antibiotic resistance gene, lentiviral LTR/Ψ packaging elements, and an sgRNA expression cassette. The precise arrangement for SKU P744452 is not provided here and should be verified against the item’s official map.

Synthetic Utility

• Not a small-molecule building block. However, in a synthetic biology context (literature/general):
  • Modular cloning: The vector can serve as a backbone for insertion or replacement of sgRNA protospacers using Golden Gate (Type IIS) cloning, Gibson assembly, or ligation-based methods.
  • Vector engineering: Swapping promoters, selection markers, or adding barcodes can tailor the construct for CRISPRi/a, base editing, or prime editing applications, provided sequence integrity is confirmed.
  • Retrosynthetic planning (conceptual): Define functional modules (ori, selection marker, sgRNA cassette, LTR/packaging signals) and choose methods to assemble or edit each module with minimal scar sequences.
  • Quality checkpoints: Post-assembly sequence verification, restriction mapping, and small-scale test transfections prior to scale-up.

Target Specificity

• Only product data may be listed here.
  • Target gene/epitope: Not specified for this item; refer to CoA/Spec Sheet.
  • Species reactivity: Not specified for this item; refer to CoA/Spec Sheet.
  • Vector backbone, promoters, selection markers, and guide sequence: Not specified for this item; refer to CoA/Spec Sheet.
  • Clone/lot information: Not specified for this item; refer to CoA/Spec Sheet.