pLenti-CBFB-sgRNA

Cat. No.: P744448

Specifiche

Product Name

pLenti-CBFB-sgRNA

Product Line

Prodotti biologici

Stoccaggio e spedizione

Condizioni di conservazione di stoccaggio

Store at -80°C

Spedito in

Dry ice packs + Cold packs

Stabilità e conservazione

Stored at -20 ℃, valid for at least 6 months;Stored at -80 ℃, valid for at least one year.

Application Protocols

No item-specific, validated protocols or recommended dilutions are provided for this product.

General workflow outlines (literature; not product-validated):

• Transfection for transient sgRNA delivery:
  • Thaw plasmid on ice, briefly spin, and keep cold. Prepare DNA–reagent complexes per transfection reagent instructions. Apply to cells at 50–80% confluence. Replace medium after 4–24 h as appropriate. Assess editing 48–96 h later.
• Lentiviral production and transduction:
  • Under BSL-2, co-transfect HEK293T with this sgRNA plasmid plus packaging/envelope plasmids. Collect and filter supernatant at 48–72 h. Determine titer. Transduce target cells at empirically determined MOI with appropriate enhancers. Select/enrich transduced cells if selectable markers are present (marker for this item is Not specified).
• Validation:
  • Extract genomic DNA; PCR-amplify the target locus; quantify editing by TIDE/ICE or amplicon NGS. Confirm functional effects with transcript/protein assays as relevant.

For detailed, step-by-step protocols, adapt manufacturer guidance from your chosen transfection reagent and packaging system. Always follow institutional biosafety policies.

Biological Roles

Target context (general, literature): CBFB (core-binding factor subunit beta) encodes a non-DNA-binding cofactor that heterodimerizes with RUNX family transcription factors, stabilizing their DNA binding and modulating transcriptional programs.

• Functional overview (literature):
  • CBFB enhances the affinity and specificity of RUNX1/2/3 for their cognate DNA elements, influencing lineage specification and differentiation programs in multiple tissues.
  • It acts as a scaffolding/regulatory subunit, impacting transcriptional initiation at promoters/enhancers where RUNX factors localize.
• Cellular pathways (literature):
  • Involved in transcriptional regulation linked to hematopoietic development and osteogenesis via RUNX partners, among other processes.
  • Modulates gene networks affecting cell cycle transitions and differentiation status in a context-dependent manner.
• Experimental implications (general):
  • Perturbing CBFB via CRISPR-mediated knockout or repression provides a route to dissect RUNX-dependent transcriptional circuits, evaluate redundancy across RUNX paralogs, and probe feedback mechanisms controlling lineage commitment.

Note: The precise sgRNA sequence, species specificity, and on/off-target profile for this item are Not specified; consult the CoA/Spec Sheet or verify empirically by sequencing-based assays.

Buffer Applications

Dedicated buffer systems (e.g., biological pH buffers for enzymology) are not a primary focus for this product. As a plasmid DNA reagent, the relevant consideration is the storage and handling buffer.

• Item-specific buffer composition: Not specified for this item; refer to CoA/Spec Sheet.
• General guidance (literature):
  • For storage, low-ionic-strength Tris (10 mM, pH ~8.0) with 0.1–1 mM EDTA can help suppress nuclease activity. Avoid EDTA for applications requiring divalent cations (e.g., some transfections or enzymatic reactions), in which case nuclease-free water or Tris without EDTA is preferred.
  • Maintain physiological ionic strength upon mixing with cell culture media to prevent osmotic stress during transfection or infection workflows.

For experimental buffers related to downstream assays (PCR, sequencing, nuclease reactions), choose validated formulations recommended by the respective enzyme manufacturers.

Green Alternatives

While plasmid DNA reagents are inherently aqueous and relatively benign, aspects of genome-editing workflows can be optimized for greener, safer practices (general, literature):

• Transfection reagents:
  • Prefer biodegradable or low-toxicity lipid-based systems over polycationic polymers with known cytotoxicity. Optimize dose to minimize waste and cell stress.
• Nucleic acid visualization:
  • Use less hazardous DNA stains (e.g., SYBR Safe-class dyes) instead of ethidium bromide for gel electrophoresis. Employ blue-light transilluminators to reduce UV exposure.
• Solvent and plastic use:
  • Minimize single-use plastics by planning aliquots. Where permitted, adopt reusable gel casting systems and reduce gel volumes. Avoid chlorinated solvents entirely (not required for DNA work).
• Energy and cold-chain:
  • Consolidate shipments and lab freezers; store at -80°C only when necessary and transition working aliquots to -20°C if compatible and validated internally (item-specific storage remains at -80°C as provided below).

Comparison (general):

• Ethidium bromide vs SYBR-type stains: SYBR-type dyes offer reduced mutagenicity and simplified waste streams, though still require proper disposal.
• PEI vs modern lipids: Lipid reagents can provide comparable transfection with lower cytotoxicity, though cost may be higher and conditions require re-optimization.

These are general recommendations; always validate alternatives in your system.

Pharmaceutical Uses

This product is for research use only and is not intended for human or animal therapeutic, diagnostic, or clinical applications.

• Excipient/formulation status: Not applicable. Plasmid DNA research reagents are not pharmacopeial excipients.
• Manufacturing context: In bioproduction research, plasmids may serve as templates for transient expression or as components in viral vector development at the research scale (general, literature). Such activities require dedicated quality systems and are out of scope for this catalog item.

Any consideration of clinical manufacturing, GMP compliance, or therapeutic use is beyond the scope of this product and is not supported.

Physical Properties

As a plasmid DNA reagent, conventional small-molecule physical constants are not typically applicable.

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Concentration/formulation: Not specified for this item; refer to CoA/Spec Sheet.
• pH of storage buffer: Not specified for this item; refer to CoA/Spec Sheet.
• Solubility: Plasmid DNA is soluble in aqueous buffers (general, literature). Organic solvent solubility is not applicable.
• Thermal behavior: DNA is thermolabile; prolonged exposure to elevated temperatures accelerates depurination and strand nicking (literature). No meaningful boiling/melting point as for small molecules; DNA exhibits cooperative strand separation dependent on GC content and ionic strength (literature).
• Optical properties (literature/general guidance):
  • Peak absorbance at 260 nm; A260 of 1.0 corresponds to ~50 µg/mL for dsDNA (literature).
  • A260/A280 ~1.8 indicates relatively pure DNA (literature). These are not specifications for this item.
• Density, logP, refractive index: Not applicable to macromolecular DNA reagents.

Note: Item-specific values (concentration, buffer composition, OD ratios) are Not specified for this item; refer to CoA/Spec Sheet.

Quality and Grades

• Item-specific grade/purity: Not specified for this item; refer to CoA/Spec Sheet.
• What quality typically means for plasmid DNA (general, literature):
  • Identity: Verified by restriction digest and/or Sanger sequencing across critical regions (promoter, sgRNA insert, junctions).
  • Integrity: Predominantly supercoiled topology assessed by agarose gel electrophoresis or capillary electrophoresis.
  • Purity: A260/A280 ~1.8 and A260/A230 >2.0 are common indicators; residual endotoxin levels may be reported when intended for sensitive cell types. These metrics are not specified for this item.
  • Functional QC: Test transfection and/or genome editing activity in a reference cell system can be included in some offerings (not specified here).
• Stabilizers/additives:
  • Not specified for this item; refer to CoA/Spec Sheet. In general, plasmids are supplied in nuclease-free water or low-salt Tris/EDTA (TE) to enhance stability (literature).
• Implications for use:
  • For lentiviral packaging or primary cell transfection, low endotoxin plasmid preparations are often preferred (literature). Confirm the endotoxin specification on the CoA if your application is sensitive.
• Documentation:
  • For sequence maps, antibiotic resistance markers, and exact sgRNA insert sequence, consult the product CoA/Spec Sheet when available. Absent explicit item data, perform incoming QC appropriate to your workflow.

Reaction and Applications

As a CRISPR sgRNA expression plasmid in a lentiviral backbone, pLenti-CBFB-sgRNA supports genome engineering workflows. The following are general, literature-based applications and practices; they are not product-specific validations.

• Typical applications (literature/general):
  • Co-transfection with a Cas9-expression plasmid or use in Cas9-expressing cells to direct double-strand breaks at CBFB target sites for gene knockout or frameshift generation.
  • Lentiviral packaging (with helper plasmids) to generate pseudotyped particles for stable delivery of the sgRNA cassette into difficult-to-transfect or dividing mammalian cells.
  • Use in CRISPRi/CRISPRa when combined with dCas9-KRAB or dCas9-activator constructs, respectively, to repress or upregulate CBFB transcription (depending on sgRNA design and TSS proximity).
• Practical considerations (general):
  • Verify the exact sgRNA sequence, PAM compatibility with the chosen Cas nuclease (e.g., SpCas9 NGG), and potential off-target sites in the host genome using in silico tools before experiments.
  • For lentiviral work, adhere to BSL-2 procedures, use third-generation packaging systems, and determine functional titer prior to large-scale experiments.
  • For editing assessment: use T7E1/TIDE/ICE analysis, amplicon NGS, or targeted qPCR; confirm protein-level effects by immunoblotting or flow cytometry where applicable.
• Controls (general):
  • Non-targeting sgRNA controls and positive-control sgRNAs against well-characterized loci are recommended to benchmark activity and specificity.

Reaction Conditions

Conventional chemical reaction conditions do not apply. The following general, literature-based conditions relate to common workflows using lentiviral sgRNA plasmids and are not item-specific recommendations:

• Transient transfection (general):
  • Cells: adherent lines such as HEK293/293T or target cell lines expressing Cas9.
  • DNA amount: optimize empirically; typical ranges 0.1–2 µg per well (6-well) depending on reagent and cell line.
  • Complexation: follow transfection reagent guidelines; maintain serum/antibiotic conditions per manufacturer.
• Lentiviral packaging (general):
  • Co-transfect with packaging and envelope plasmids in HEK293T under BSL-2 conditions.
  • Harvest supernatant 48–72 h post-transfection; clarify and, if needed, concentrate by PEG precipitation or ultracentrifugation.
  • Determine functional titer before transduction; polybrene usage and spinoculation can enhance efficiency but require cell-type optimization.
• Editing assessment (general):
  • Sample genomic DNA 48–96 h post-delivery for early editing readouts; for clonal lines, expand single cells and validate by sequencing.

These conditions are intended as high-level guidance. Optimize parameters for your specific cell type, delivery method, and biosafety requirements. No product-specific performance data are provided.

Safety and Handling

• GHS/SDS information:
  • Signal word, hazard statements, pictograms: Not specified for this item; refer to SDS.
  • Plasmid DNA in aqueous buffer is generally considered non-hazardous chemically; always follow institutional biosafety procedures.
• Biosafety context (general, literature):
  • This product is a DNA plasmid. It is non-infectious by itself. Packaging into lentiviral particles requires co-transfection with helper plasmids; such work is typically conducted under BSL-2 containment following institutional approvals.
• Personal protective equipment (PPE):
  • Laboratory coat, disposable nitrile gloves, and eye protection when handling solutions and during transfection/packaging workflows.
• Handling practices:
  • Work on ice for prolonged manipulations to minimize nuclease activity.
  • Use nuclease-free plastics and solutions. Avoid repeated freeze–thaw cycles to limit nicking and degradation.
  • Prevent aerosol generation; disinfect work surfaces after use.
• Incompatibilities and stability:
  • Avoid strong acids/bases, oxidizers, nucleases, and high temperatures. Do not autoclave the DNA solution.
• First-aid (general):
  • Skin/eye contact with buffer: rinse with water. Ingestion/inhalation: seek medical attention per institutional policy. Defer to SDS for authoritative guidance.
• Waste disposal:
  • Dispose of DNA-containing materials and cell culture waste in accordance with local biosafety and institutional guidelines. Decontaminate with appropriate disinfectants (e.g., 10% bleach) for surfaces and liquid waste as required.

Solvent Selection

This product is an aqueous DNA reagent; conventional organic solvent selection does not apply.

• Applicable media (general, literature):
  • Nuclease-free water for dilution/short-term use when compatible with downstream applications.
  • Low-ionic-strength buffers (e.g., 10 mM Tris-HCl, pH ~8.0, with or without 0.1–1 mM EDTA) to enhance long-term stability by chelating divalent cations required for nuclease activity. Specific buffer for this item is Not specified; refer to CoA/Spec Sheet.
• Miscibility: Fully miscible with aqueous buffers and cell culture media; avoid exposure to organic solvents which can denature or precipitate DNA (literature).
• When to choose water vs TE (general guidance):
  • Choose nuclease-free water for immediate transfection or enzyme reactions sensitive to EDTA.
  • Choose TE for storage/transport to protect from nuclease degradation.

For experimental design, prioritize the sections on Reaction & Applications and Storage & Reconstitution rather than solvent choice.

Storage and Reconstitution

• Storage conditions (item-specific): Store at -80°C. Avoid repeated freeze–thaw cycles by preparing working aliquots.
• Shipping (item-specific): Shipped on dry ice packs + cold packs to maintain the frozen/cold chain.
• Reconstitution/Dilution: Not specified for this item; refer to CoA/Spec Sheet. General guidance (literature): If supplied lyophilized (not specified), reconstitute in nuclease-free water or low-salt Tris buffer to the desired concentration. If supplied in solution, thaw on ice, mix gently (do not vortex vigorously), briefly spin down, and keep on ice during setup.
• Handling notes (general):
  • Use sterile, nuclease-free tips and tubes. Work quickly on ice to minimize nuclease activity. Record the number of freeze–thaw events.
  • Store long-term at -80°C in small, single-use aliquots. For short-term work (days to a week), -20°C may be acceptable for many plasmids, but the specified storage for this item remains -80°C.
• Stability: Specific shelf-life and buffer composition are Not specified for this item; refer to CoA/Spec Sheet. Validate plasmid integrity by agarose gel or sequencing after extended storage.
• Research use note: For research use only.

Structure and Identity

pLenti-CBFB-sgRNA is a recombinant DNA plasmid designed to express a single guide RNA (sgRNA) targeting the CBFB gene within a lentiviral backbone for CRISPR workflows.

• Item-specific identifiers:
  • CAS: Not specified for this item; refer to CoA/Spec Sheet.
  • 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/InChIKey: Not applicable to large DNA constructs; Not specified for this item; refer to CoA/Spec Sheet.
• Structural features (general, literature):
  • Circular, double-stranded DNA plasmid (supercoiled form typically supplied for transfection and cloning).
  • Lentiviral backbone enabling packaging into pseudotyped viral particles when co-transfected with helper plasmids (literature).
  • sgRNA expression cassette commonly driven by a U6 or comparable RNA polymerase III promoter (literature; promoter for this item not specified).
  • May contain bacterial selection marker and origin of replication for propagation in E. coli (literature; specific elements for this item not specified).
• 2D structural description (general):
  • Topologically circular dsDNA with modular cassettes arranged as promoter – sgRNA scaffold – terminator; auxiliary modules include antibiotic resistance and ori. Exact map and sequence for this item are not specified; consult the product CoA/Spec Sheet if available.

Synthetic Utility

Traditional small-molecule synthetic transformations do not apply to plasmid DNA. However, from a molecular biology perspective, this vector provides modular utility in genetic construct assembly (general, literature):

• Cloning utility:
  • The sgRNA cassette can be re-targeted by ligating alternative spacer sequences using restriction/ligation or Golden Gate cloning, enabling rapid retargeting to new loci.
  • Backbone features (promoters, selection markers) can be swapped using Gibson Assembly or other homologous recombination methods to tailor expression contexts.
• Retrosynthetic viewpoint (construct design):
  • Decompose desired function into modules: delivery (lentiviral elements), expression (U6/other Pol III promoter), selection (antibiotic or fluorescent marker), and targeting (20-nt spacer). Each module can be redesigned independently.
• Downstream enablement:
  • Serves as a component in multigene delivery systems (with Cas9, reporters) and facilitates stable or transient modulation of the CBFB locus in mammalian cells.

Note: Specific restriction sites, overhangs, and sequence information for this item are Not specified; refer to CoA/Spec Sheet or map the plasmid by sequencing.

Target Specificity

• Stated target based on product name: CBFB. The exact spacer sequence, PAM compatibility, and species ortholog specificity are Not specified for this item; refer to CoA/Spec Sheet.
• Off-target/ontarget characterization: Not specified for this item; users should validate specificity in their system via sequencing-based assays (general, literature).

No additional antigen/epitope or clone information is provided; this product is a plasmid, not an antibody.