pLenti-CBFB-sgRNA

Cat. No.: P744447

사양

Product Name

pLenti-CBFB-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 3개월 유효, -80℃에서 보관하면 1년 이상 유효합니다.

Application Protocols

Only provide item-specific, validated application details here.

• Tested applications (e.g., WB, IHC, IF, FC for antibodies; or specific transfection/transduction protocols for vectors) and recommended conditions: Not specified for this item; refer to CoA/Spec Sheet.

Manufacturer Applications text was not provided beyond the product name; no additional, product-validated protocols are available in the Product Data.

Biological Roles

About CBFB (general biology; not product specifications):

• CBFB (core-binding factor subunit beta) encodes a non-DNA-binding cofactor that heterodimerizes with RUNX transcription factors to increase their DNA-binding affinity and stability. The RUNX–CBFβ complex regulates transcriptional programs in hematopoiesis, osteogenesis, and other developmental processes.
• Mechanistic role: CBFβ stabilizes the Runt domain of RUNX proteins on target DNA consensus motifs, influencing chromatin recruitment of co-regulators. Modulation of CBFB levels can alter lineage specification and transcriptional output of RUNX-dependent genes.
• Cellular pathways: Implicated in regulation of cell cycle–related genes, differentiation markers, and components of signaling cascades where RUNX factors act as integrators.

Relevance to this plasmid (context):

• An sgRNA targeting CBFB enables perturbation of the RUNX/CBF transcriptional axis for fundamental research, such as dissecting transcriptional networks, identifying genetic dependencies, or validating pathway interactions in cell-based models.

Note: The above reflects literature/general knowledge about gene function. This product is for research use only and is not intended for diagnostic or therapeutic applications.

Buffer Applications

Not a conventional buffer reagent. However, plasmid DNA stability and performance depend on the surrounding buffer system (general guidance):

• Long-term storage: TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5–8.0) helps protect DNA from nucleases via EDTA chelation of Mg2+. For applications sensitive to EDTA (e.g., certain transfections or Mg2+-dependent enzymes), store in low-EDTA TE (e.g., 10 mM Tris, 0.1 mM EDTA) or nuclease-free water and use promptly.
• Working solutions: Dilute in nuclease-free water or appropriate reaction buffers (e.g., restriction enzyme buffers, ligation buffers) as required by the protocol.
• Electrophoresis: Analyze plasmid integrity on 0.7–1% agarose gels in TAE or TBE buffer; supercoiled, nicked, and linear forms migrate distinctly.

Item-specific buffer composition is not specified for this product; refer to the CoA/Spec Sheet.

Green Alternatives

While “solvent” green alternatives are not directly applicable to plasmid DNA, greener practices can be implemented around its use (literature/general guidance):

• DNA-based editing vs. chemical mutagenesis: CRISPR plasmid approaches avoid hazardous mutagens (e.g., EMS), reducing toxic waste generation. Tradeoff: molecular biology plastics and single-use consumables increase solid waste; implement recycling programs where permitted.
• Endotoxin-reduced prep methods: Use silica membrane or magnetic bead kits that minimize chaotropic salt usage compared with traditional phenol–chloroform extraction. Tradeoff: cartridge waste vs. reduced organic solvent hazards.
• Aqueous-only workflows: Favor water/TE buffers and avoid phenol/chloroform where possible; use enzyme-based cleanup (e.g., exonuclease treatments) as alternatives to solvent extraction.

Comparison (general):

• Traditional plasmid purification (phenol/chloroform): High purity, but hazardous solvents and disposal burden.
• Modern kit-based purification (silica/beads): Comparable purity for most applications, minimal hazardous solvent use, easier waste handling.

Pharmaceutical Uses

This product is a research-use-only plasmid and is not intended for human or veterinary use, diagnostic procedures, or as a drug substance or excipient.

Formulation/manufacturing context (general information):

• Plasmid DNA used in pharmaceutical manufacturing (e.g., as template for RNA production or as an intermediate in vector manufacture) is typically controlled under stringent quality systems (endotoxin, residual host contaminants, sequence identity). Such qualifications are not specified for this item; refer to the CoA/Spec Sheet if applicable.
• No pharmacopeial monograph applies to this research plasmid.

Physical Properties

Item-specific physical properties:

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Concentration, solvent/buffer, A260/280: Not specified for this item; refer to CoA/Spec Sheet.
• Density, refractive index, pKa/logP: Not applicable/not specified for plasmid DNA; refer to CoA/Spec Sheet.

General properties (literature/general knowledge for plasmid DNA):

• State: Nucleic acid supplied typically in aqueous, nuclease-free buffer (e.g., water or TE). Plasmid DNA is highly water-soluble; insoluble in nonpolar organic solvents.
• Thermal behavior: Double-stranded DNA exhibits cooperative melting (Tm) that depends on length, GC content, and ionic strength; supercoiled plasmids denature at higher temperatures than linear fragments. Avoid prolonged heating >65 °C unless intentionally denaturing.
• UV characteristics: Strong absorbance at 260 nm; A260 of 1.0 corresponds to ~50 µg/mL for dsDNA (literature). A260/280 of ~1.8 indicates low protein contamination.
• Stability: Sensitive to nucleases (DNase I, restriction enzymes) and to repeated freeze–thaw; stable for months at −20 to −80 °C in TE buffer (literature).

Quality and Grades

Item-specific quality information:

• Grade/Purity: Not specified for this item; refer to CoA/Spec Sheet.

How to interpret quality for plasmid DNA (general guidance):

• Common quality attributes for plasmids include supercoiled percentage, A260/280 (~1.8) and A260/230 (>2.0) ratios, residual endotoxin (EU/µg DNA), residual genomic DNA/RNA, and sequence verification of key cassettes (e.g., sgRNA spacer).
• Research Grade vs. Transfection Grade: Research-grade plasmids are suitable for routine cloning and PCR. Transfection-grade or endotoxin-reduced plasmids are preferred for sensitive mammalian cell transfections or in vivo animal research. Unless explicitly stated, assume research-use only.
• Sequence fidelity: For sgRNA vectors, verify the 20-nt spacer sequence, absence of frameshifts/indels in the scaffold, and integrity of promoter and LTR regions by Sanger or NGS as appropriate.
• Function checks: A quick-cut restriction digest to confirm plasmid size and a test PCR across the sgRNA insert provide practical verification prior to large-scale experiments.
• Documentation: For item-specific identity, concentration, and recommended use, consult the CoA/Spec Sheet. Do not infer endotoxin levels, cassette details, or antibiotic markers without documentation.

Reaction and Applications

Manufacturer Applications: Not specified for this item in the Product Data.

Typical applications for pLenti sgRNA plasmids (literature/general knowledge):

• CRISPR workflows: Expression of a CBFB-targeting sgRNA for CRISPR/Cas9-based gene knockout, repression (CRISPRi with dCas9-KRAB), or activation (CRISPRa with dCas9-VP64) when co-delivered with the appropriate Cas9/dCas9 construct.
• Lentiviral delivery: Packaging into replication-incompetent lentiviral particles for stable delivery to dividing and non-dividing mammalian cells. Co-transfect with compatible packaging plasmids (e.g., gag/pol and VSV-G) into producer cells such as HEK293T; transduce target cells at controlled MOI.
• Cloning/engineering: Spacer replacement via BsmBI/BbsI Golden Gate cloning is common for sgRNA vectors; verify insertion by colony PCR and Sanger sequencing.
• Functional genomics: Use in pooled or arrayed screens targeting CBFB or CBFB-dependent pathways, with readouts such as NGS, qPCR, or reporter assays.

Practical tips:

• Maintain sterility and nuclease-free conditions; verify plasmid map and antibiotic resistance before transformation.
• For lentiviral work, titer normalization (e.g., RT-qPCR p24 surrogate) and antibiotic selection of transduced cells are standard; choose selection markers compatible with the destination cells. All such steps should follow institutional biosafety rules.

Reaction Conditions

Item-specific reaction conditions are not provided in the Product Data. The following are general, literature-based guidelines for typical workflows using lentiviral sgRNA plasmids:

• Transfection (producer cells): HEK293T cells are commonly transfected with the transfer plasmid plus packaging (gag/pol) and envelope (e.g., VSV-G) plasmids using PEI or lipid-based reagents. Maintain cells at 37 °C, 5% CO2; change media after 6–16 h if required by reagent; harvest viral supernatant at 48–72 h post-transfection.
• Transduction (target cells): Add filtered viral supernatant with polybrene (if compatible) to target cells; spinoculation can increase efficiency. Choose MOI according to single-copy vs. multi-copy goals. Replace media after 12–24 h and begin antibiotic selection if the vector contains a resistance marker.
• CRISPR editing: Co-deliver Cas9 (plasmid, RNP, or stable) with the sgRNA vector. Editing outcomes are typically assessed 48–96 h post-delivery via T7E1/Surveyor assays, amplicon NGS, or targeted qPCR.
• Controls: Include a non-targeting sgRNA and a positive-control sgRNA against a known essential locus to benchmark performance.

Always optimize reagent ratios, cell density, and timing for each cell line. Follow institutional biosafety policies for handling lentiviral materials.

Safety and Handling

Item-specific safety data:

• GHS Classification, Signal Word, Pictograms, H-Statements: Not specified for this item; refer to SDS.

General guidance (literature/biosafety best practice):

• This product is a plasmid DNA preparation for research use only. Plasmid DNA itself is not infectious; however, if used to generate lentiviral particles, handle downstream lentiviral preparations under appropriate institutional biosafety approvals (typically BSL-2 with enhancements per local EHS guidance).
• PPE: Lab coat, disposable nitrile gloves, and safety glasses. Use nuclease-free technique to avoid degradation (clean benches/equipment, use filtered tips, dedicated nuclease-free reagents).
• Engineering controls: Perform manipulations that may generate aerosols (e.g., vortexing viral supernatants, spin-filtration) in a certified biological safety cabinet if virus is produced. For routine plasmid handling, standard chemical fume hoods are not required.
• Incompatibilities: Avoid contaminating with nucleases or strong oxidants; avoid exposure to UV light which can damage nucleic acids.
• First aid (general): In case of accidental exposure to buffer/plasmid solution, rinse skin/eyes with water. If ingested or inhaled as aerosol, seek medical advice and provide SDS.
• Decontamination: Surfaces contacted by DNA can be cleaned with 10% bleach followed by ethanol/water. For lentiviral materials, use freshly prepared 10% bleach with appropriate contact time and dispose as biohazard waste.
• Disposal: Follow institutional biosafety procedures for GMO-containing waste. Consult SDS for authoritative information.

Solvent Selection

This section is not typically applicable to plasmid DNA as a reagent. The product is generally supplied in an aqueous, nuclease-free buffer and is intended to be handled in water-based solutions.

General guidance (literature):

• Preferred solvents: Nuclease-free water or TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5–8.0) for long-term stability; low-EDTA or water for applications sensitive to chelators (e.g., transfection with certain reagents, Mg2+-dependent enzymes).
• Avoid: Nonpolar organic solvents (immiscible, denaturing), highly acidic/basic conditions that hydrolyze phosphodiester bonds, and solutions contaminated with nucleases.
• Additives: For some workflows, adding carrier (e.g., low concentrations of glycogen) can aid precipitation; use only if compatible with downstream steps.

Storage and Reconstitution

Item-specific storage and shipping (from Product Data):

• Storage Conditions: Store at −80 °C.
• Shipped In: Dry ice packs + Cold packs.
• Research Use Note: For research use only.

General handling and reconstitution (literature/best practice for plasmid DNA):

• Upon receipt: Keep frozen. Minimize time at ambient temperature. Thaw on ice, mix gently by slow pipetting; avoid vigorous vortexing that can shear DNA.
• Aliquoting: If repeated use is anticipated, aliquot into nuclease-free tubes to avoid freeze–thaw cycles. Return unused material promptly to −80 °C. For short-term storage (days to weeks), −20 °C is commonly acceptable; long-term per item data is −80 °C.
• Buffer: If concentration adjustment is needed, dilute with nuclease-free water or TE buffer (pH 7.5–8.0). For applications sensitive to EDTA, use water or low-EDTA buffers and keep on ice during setup.
• Stability: Avoid repeated freeze–thaw and nuclease contamination. Store away from light to prevent UV-induced damage.
• Documentation: For any item-specific concentration, buffer composition, and recommended working dilutions, consult the CoA/Spec Sheet.

Do not deviate from the specified −80 °C storage without internal validation, and always handle according to institutional biosafety policies when using the plasmid to generate lentiviral particles.

Structure and Identity

Item-specific identifiers (from Product Data):

• CAS: Not specified for this item; refer to CoA/Spec Sheet.
• InChIKey: Not applicable to plasmid DNA; not assigned.
• SMILES: Not applicable to plasmid DNA; not assigned.
• Molecular Formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular Weight: Not specified for this item; refer to CoA/Spec Sheet.

General structural description (literature/general knowledge):

• pLenti-CBFB-sgRNA is a recombinant DNA plasmid in a lentiviral backbone engineered to express a single-guide RNA (sgRNA) directed to the CBFB locus. As a plasmid, it is a covalently closed, circular, double-stranded DNA molecule.
• Typical functional modules in pLenti sgRNA vectors include: a U6 (or other Pol III) promoter driving the sgRNA scaffold with a 20-nt spacer sequence; a bacterial origin of replication (e.g., ColE1) and antibiotic resistance cassette for E. coli propagation; and lentiviral long terminal repeats (LTRs) with packaging signal (Ψ) for production of replication-incompetent lentiviral particles. Exact sequence features for this item are not specified here.
• 2D/functional map in words: bacterial propagation region (ori + selection marker) → lentiviral transfer cassette bracketed by LTRs → U6 promoter → gRNA scaffold containing the CBFB-targeting spacer.
• Stereochemistry: Not applicable (polymeric nucleic acid).

Synthetic Utility

From a synthetic biology perspective (literature/general knowledge):

• Template utility: Serves as a template for PCR amplification of the sgRNA cassette or for in vitro transcription of gRNA when paired with a T7 promoter-bearing construct.
• Modular cloning: The sgRNA spacer region in pLenti backbones is commonly exchanged by Golden Gate cloning using type IIS restriction enzymes (e.g., BsmBI/BbsI), enabling rapid generation of alternate CBFB-targeting sequences or controls.
• Vector engineering: Useful as a donor for subcloning the U6–sgRNA cassette into alternative vectors (e.g., non-viral plasmids, AAV transfer plasmids) via Gibson Assembly, In-Fusion, or classical ligation.
• Barcoding and screening: Can be adapted for pooled screening by incorporating unique molecular identifiers or incorporating compatible selection markers/reporters in the lentiviral cassette.
• Retrosynthetic perspective: The functional unit is the sgRNA spacer+scaffold under a small RNA promoter; upstream/downstream modules (selection marker, promoter, LTRs) can be treated as interchangeable blocks depending on delivery route and selection strategy.

Note: Exact restriction sites, selection markers, and cassette details for this specific item are not specified; confirm with the plasmid map/CoA before designing cloning steps.

Target Specificity

Only provide item-specific details here.

• Target antigen/gene, species reactivity, isotype/clone (if antibody), epitope: Not specified for this item; refer to CoA/Spec Sheet.

Note: Product name indicates an sgRNA directed to CBFB, but the exact spacer sequence, isoform targeting, and species context are not specified in the Product Data.