pLenti-U2AF1-sgRNA

Cat. No.: P751464

Specifications

Product Name

pLenti-U2AF1-sgRNA

Product Line

생물 제품

보관 및 배송

보관 조건

Store at -80°C

배송

Dry ice packs + Cold packs

안정성 및 스토리지

Stored at -20 ℃, valid for 3 months;Stored at -80 ℃, valid for at least one year.

Application Protocols

No tested applications, protocols, or recommended conditions are specified in the Product Data for this item.

Please refer to the product CoA/Spec Sheet and your institution’s standard operating procedures for detailed, validated methods relevant to pLenti-based sgRNA vectors. General protocols for cloning, transfection, lentiviral packaging, transduction, and editing validation are widely available in the literature and from core facilities.

Biological Roles

Item context: The product name indicates an sgRNA directed toward U2AF1, a splicing factor. No target sequence or validation data are provided for this SKU.

General biology of U2AF1 (literature; not item-specific):

• U2AF1 (also known as U2AF35) is the small 35-kDa subunit of the U2 auxiliary factor heterodimer (U2AF1/U2AF2) essential for pre-mRNA splicing.
• Function: Recognizes the AG dinucleotide at the 3′ splice site and coordinates recruitment of U2 snRNP to the branch point, facilitating spliceosome assembly.
• Domains/features: Contains CCCH-type zinc fingers and an RS domain mediating RNA binding and protein–protein interactions within the spliceosome.
• Cellular roles: Impacts alternative splicing decisions; perturbation can alter exon inclusion/exclusion and transcript isoform profiles.
• Research significance: U2AF1 perturbation is used to probe splicing fidelity, exon definition mechanisms, and the coupling between splicing and transcription/translation. Editing or knockdown experiments often measure changes by RNA-seq, RT–qPCR splicing assays, and proteomics.

Caveats for CRISPR targeting (general):

• U2AF1 is an essential gene in many cell contexts; strong loss-of-function may reduce viability. Consider inducible systems, partial knockdown strategies, or CRISPRi for tunable repression.
• Validate on-target effects at both genomic and functional levels (e.g., splicing reporter assays), and assess potential compensation by splicing network factors.

Buffer Applications

This product is a plasmid DNA reagent; classical biological buffer applications (e.g., running buffers for electrophoresis or cell-culture media) are not the primary focus.

General practice for plasmid DNA buffering (literature; not item-specific):

• Long-term storage: TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) is widely used to maintain DNA integrity by buffering pH and chelating Mg2+ that nucleases require.
• Working aliquots: Nuclease-free water or low-ionic Tris can be used for immediate transfection or enzyme reactions. Avoid EDTA in reactions requiring Mg2+ (e.g., polymerases, ligases) unless appropriately diluted.
• Electrophoresis: TAE or TBE buffers are standard for analytical gels when verifying plasmid integrity or insert size.
• Sterility: Filter-sterilize buffers and use DNase/RNase-free consumables to prevent degradation.

Note: Exact buffer composition for SKU P751464 is not specified; refer to the CoA/Spec Sheet.

Green Alternatives

Sustainability considerations for genome-editing workflows involving lentiviral sgRNA vectors:

Greener or lower-risk alternatives (general literature comparison; not item-specific):

• RNP delivery (Cas9 protein + synthetic sgRNA):
  • Pros: No plasmid backbone; transient; minimizes off-target duration; avoids viral packaging and associated biohazard waste.
  • Cons: Higher per-reaction cost; transient effect may limit long-term studies without selection.
• Non-viral plasmid delivery (electroporation, lipid-mediated):
  • Pros: Avoids generation of viral waste; simpler compliance; reduced PPE requirements versus BSL-2 viral work.
  • Cons: Lower delivery efficiency in some primary cells; transient unless integrated systems are used.
• Self-inactivating vectors with reduced antibiotic markers:
  • Pros: Decreases selection pressure in environmental release scenarios; simplifies downstream purification.
  • Cons: Feature-dependent; may limit selectable markers.

Small comparison (literature):

• Lentiviral sgRNA plasmid: Highest versatility and stable integration; requires BSL-2, bleach decontamination, and plastic waste from viral work.
• RNP editing: Minimal waste and rapid workflows; requires cold-chain for protein and synthetic RNA.

Operational tips to “green” your process:

• Use miniaturized transfections, high-efficiency reagents to reduce DNA and plastic use.
• Consolidate bleach decontamination, and neutralize properly to minimize corrosive waste.
• Plan aliquots to avoid freeze–thaw loss and repeat orders, reducing shipping footprint.

Note: These are general sustainability perspectives; select methods based on scientific requirements and institutional policies.

Pharmaceutical Uses

Not applicable for clinical use.

Item-specific statement:

• Research Use Only: This product is supplied exclusively for research purposes and is not intended for human or veterinary use, diagnostics, or as a drug substance/excipient in manufacturing.

General context (not item-specific):

• In pharmaceutical R&D laboratories, genome editing tools may be used for discovery biology, target validation, and cell line engineering. When plasmid or viral vectors are used in GMP environments, specialized GMP-grade materials, facility segregation, and extensive documentation are required.
• Transitioning from research-grade to clinical-grade vectors involves comprehensive quality attributes (sequence confirmation, sterility, endotoxin, residual host contaminants, replication-competent virus testing for lentiviral products). None of these clinical-grade attributes are specified or implied for this SKU.

Conclusion:

• No pharmacopeial status or formulation role is claimed for pLenti-U2AF1-sgRNA. Use only in preclinical research following institutional biosafety policies.

Physical Properties

Item-specific data:

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Concentration, A260/A280, endotoxin level, and buffer composition: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight (plasmid length): Not specified for this item; refer to CoA/Spec Sheet.

General/literature properties for plasmid DNA reagents (not item-specific):

• Physical state: Aqueous solution (commonly in nuclease-free water or TE) or lyophilized DNA. Highly water-soluble.
• Thermal behavior: No conventional melting/boiling points; double-stranded DNA exhibits a cooperative thermal denaturation (Tm) dependent on length, GC content, and ionic strength.
• Density/refractive index: Governed by the aqueous buffer; DNA contributes minimally at typical microgram-per-mL concentrations.
• pH considerations: DNA is most stable near neutral to slightly basic pH (e.g., TE pH ~8.0 helps limit depurination).
• Solubility: Readily soluble in common biological buffers (water, TE, Tris, PBS); insoluble in most organic solvents.
• Stability factors: Sensitive to nucleases, repeated freeze–thaw, extreme pH (<6 or >10), and elevated temperatures. Chelation of divalent cations (e.g., 1 mM EDTA) helps mitigate nuclease activity.

Note: For exact plasmid length, sequence map, concentration, and buffer for SKU P751464, consult the CoA/Spec Sheet.

Quality and Grades

Item-specific grade/purity: Not specified for this item; refer to CoA/Spec Sheet.

How to interpret grades for DNA/vector reagents (general, not item-specific):

• Research-grade plasmid DNA is commonly provided at high purity suitable for routine cloning, transfection, and virus packaging. Typical controls include OD260/280 and agarose gel integrity. Endotoxin-reduced or “transfection-grade” DNA is often preferred for sensitive cell types and for lentiviral production to maximize titer.
• Sequence identity is commonly verified by restriction analysis and/or Sanger/NGS of the sgRNA cassette and vector backbone junctions. Exact verification methods for this SKU are not specified.
• Low UV absorbance contaminants (phenol, guanidine) and high endotoxin can reduce transfection efficiency and cell viability; premium grades address these through purification workflows.

What is known for this item:

• Research use only. No clinical or diagnostic use is implied.
• Storage requirement is specified at −80°C (see Storage & Reconstitution tab). Other quality metrics (concentration, endotoxin, host strain, antibiotic marker) are not specified; consult the CoA/Spec Sheet for batch-specific data.

Practical note:

• If you intend to package lentivirus or transfect primary cells, consider requesting or preparing endotoxin-minimized DNA. Validate identity by sequencing the sgRNA region before critical experiments.

Reaction and Applications

Item context: pLenti-U2AF1-sgRNA is intended for CRISPR workflows where an sgRNA against U2AF1 is expressed from a lentiviral plasmid. Specific cassette design and sequence are not provided in the Product Data.

General applications for pLenti sgRNA vectors (literature guidance):

• Stable gene perturbation:
  • Knockout with Cas9 by inducing DSBs at the U2AF1 locus, leading to indels via NHEJ.
  • CRISPRi/CRISPRa when paired with dCas9-KRAB or dCas9-activator systems and sgRNAs targeting promoter/TSS regions.
• Lentiviral delivery:
  • Enables transduction of dividing and non-dividing cells; integration supports long-term expression. Use replication-incompetent packaging systems.
• Library/pooled screens:
  • U2AF1-targeting constructs can be included in focused splicing-factor libraries; maintain representation with low MOI and antibiotic selection.

Practical tips (general):

• Verify sgRNA spacer sequence by Sanger sequencing prior to critical experiments.
• For packaging: Co-transfect packaging plasmids (e.g., psPAX2, pMD2.G or equivalents) into HEK293T; harvest 48–72 h. Filter (0.45 µm) and optionally concentrate.
• Use appropriate controls: non-targeting sgRNA, safe-target controls, and known on-target controls.
• Validate editing: TIDE/ICE, amplicon NGS, or locus-specific assays; confirm at the protein or functional level where relevant.

Note: Exact applications validated for SKU P751464 are not specified; refer to the product’s CoA/Spec Sheet and your institutional SOPs.

Reaction Conditions

No item-specific, validated experimental conditions are provided for SKU P751464.

General literature guidance for common workflows with lentiviral sgRNA plasmids (not item-specific; adjust per lab SOPs):

• Transient transfection (HEK293T for packaging):
  • DNA quality: Endotoxin-reduced, A260/280 ~1.8–2.0.
  • Reagents: PEI, lipofection reagents, or calcium phosphate. Optimize DNA:reagent ratios per manufacturer guidance.
  • Timing: Harvest viral supernatant at 48 and 72 h post-transfection; filter (0.45 µm). Optional PEG/ultracentrifugation concentration.
• Cell transduction:
  • MOI: Use low MOI (<0.3) for pooled screens; adjust for single-copy integration. Include polybrene if compatible; spinoculation may improve efficiency.
  • Selection: Begin antibiotic selection 48–72 h post-transduction if the vector encodes a selectable marker; duration typically 3–7 days until mock-transduced cells are eliminated.
• Editing validation:
  • Genotyping: Amplicon sequencing, T7E1/Surveyor assays, or ICE/TIDE analysis 3–10 days after editing.
  • Functional assays: RNA-seq or RT–qPCR for splicing changes when perturbing U2AF1.

Environmental/sterility parameters:

• Perform viral work at BSL-2 with appropriate containment.
• Maintain nuclease-free technique for plasmid handling.

These conditions are general starting points; fine-tune for your cell type, delivery platform, and institutional policies.

Safety and Handling

Item-specific hazard information:

• Signal word, H-statements, GHS classification, pictograms: Not specified for this item; refer to SDS.
• Research Use Note: For research use only.

General safety guidance for lentiviral sgRNA plasmids and their use (not item-specific):

• Plasmid DNA itself is typically low hazard; however, when used to generate lentiviral particles, handle all steps under appropriate biosafety level (commonly BSL-2) with institutional approvals.
• Avoid aerosol generation. Work in a certified Class II biosafety cabinet during virus production, concentration, and handling. Decontaminate surfaces with freshly prepared 10% bleach followed by 70% ethanol.
• Use appropriate PPE: lab coat, gloves, and eye protection. Change gloves frequently to prevent nuclease contamination.
• Incompatibilities: DNA is degraded by nucleases; keep solutions and tools nuclease-free. Avoid repeated freeze–thaw; aliquot as needed.
• First aid (general): If exposure to transfection reagents/viral supernatants occurs, rinse affected area with water; seek medical attention per institutional policy. Report potential exposures to biosafety officer.
• Waste: Deactivate lentiviral waste by bleach treatment or validated chemical/thermal methods before disposal, per local regulations.
• Dry ice and cold-pack shipping: Handle dry ice with cryo-gloves; allow packages to equilibrate briefly before opening to prevent condensation.

Always defer to the SDS, institutional biosafety guidelines, and your IBC-approved SOPs for authoritative safety procedures.

Solvent Selection

This product is a plasmid DNA reagent, not a solvent; conventional solvent selection does not apply.

General handling media for plasmid DNA (literature guidance, not item-specific):

• Common resuspension/storage buffers:
  • Nuclease-free water (pH ~7–8): maximizes compatibility for downstream enzymatic reactions; may reduce long-term stability compared to buffered solutions.
  • TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0): widely used for stability; EDTA chelates Mg2+ to inhibit nucleases. For immediate transfection, dilution into salt-free water or transfection buffer can be performed.
  • Tris buffer without EDTA: sometimes preferred when immediate use in Mg2+-dependent reactions is planned.
• Avoid organic solvents (e.g., alcohols, acetone, chlorinated solvents) which precipitate or denature DNA.

Selection considerations:

• For cloning or enzyme digestion: Use low-salt, nuclease-free water or TE; adjust ionic strength per enzyme requirements.
• For transfection: Many reagents prefer DNA in water or low-ionic buffers; follow reagent-specific recommendations.
• For lentiviral packaging: High-purity, endotoxin-reduced DNA in water or low-salt buffer improves complexation with transfection reagents (PEI, lipids).

Storage and Reconstitution

Item-specific instructions:

• Storage conditions: Store at −80°C.
• Shipping: Shipped on dry ice packs + cold packs.

Additional general guidance (literature; not item-specific):

• Upon receipt: Minimize freeze–thaw cycles. Centrifuge briefly to collect contents. Keep on ice during handling.
• Aliquoting: Divide into small, single-use aliquots in nuclease-free tubes to avoid repeated freeze–thaw. Label with concentration, buffer, and date.
• Short-term storage: For frequent use, −20°C storage may be acceptable for aliquots; for long-term integrity, maintain a master stock at −80°C.
• Buffer considerations: TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) enhances DNA stability. If the product is supplied in water, consider adding Tris/EDTA when long-term storage is required, unless immediate use in Mg2+-dependent reactions is planned.
• Thawing: Thaw on ice. Mix gently by pipetting; avoid vigorous vortexing to prevent shearing of high-MW DNA.
• Sterility and nuclease control: Use DNase/RNase-free tips and tubes; keep work surfaces clean. Avoid contact with skin or non-sterile surfaces.

Note: If reconstitution (from dry or lyophilized form) were required, use nuclease-free water or TE to the desired concentration. For this SKU, specific appearance/formulation is not specified; refer to the CoA/Spec Sheet.

Structure and Identity

Brief description: pLenti-U2AF1-sgRNA is a lentiviral vector reagent intended for CRISPR research workflows where an sgRNA directed at the human U2AF1 gene is expressed from a plasmid backbone.

Item-specific facts (from Product Data):

• SKU: P751464
• Product name: pLenti-U2AF1-sgRNA
• 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 / InChI / InChIKey: Not applicable for a plasmid DNA construct; not specified for this item.

General/literature description of structure (not item-specific):

• A “pLenti” sgRNA vector typically consists of:
  • 5′ and 3′ lentiviral LTRs and a Ψ packaging signal enabling production of replication-incompetent lentiviral particles in packaging cells.
  • A Pol III promoter (commonly human U6) driving expression of the sgRNA spacer fused to the standard tracrRNA scaffold.
  • A bacterial origin of replication (e.g., ColE1) for propagation in E. coli and an antibiotic resistance marker for plasmid selection (e.g., AmpR or KanR).
  • Optional mammalian selection marker (e.g., puromycin) and/or fluorescent reporter for tracking transduction. Specific features for this item are not specified.

2D/functional features (literature):

• Linear sequence of double-stranded DNA comprising functional modules: LTR–Ψ–(promoters/cassettes)–selection marker–origin. The sgRNA region contains a 20-nt spacer (sequence not provided here) upstream of the invariant tracr scaffold.

Synthetic Utility

Classical small-molecule synthetic utility does not apply to a plasmid DNA reagent. However, from a molecular biology engineering perspective, the vector backbone has utility as a cloning scaffold.

General vector engineering utility (literature; not item-specific):

• sgRNA cassette exchange: Many pLenti sgRNA vectors allow spacer swap via BsmBI/BbsI Golden Gate cloning or Gibson assembly, enabling rapid retargeting to different loci.
• Modular design: Promoter, selection marker, and reporter modules can be replaced through standard restriction/ligation or sequence-assembly methods to tailor expression or selection conditions.
• Multiplexing: Tandem sgRNA cassettes or Csy4/tRNA processing systems can be inserted for multi-site targeting.
• Compatibility: Co-expression with Cas9 (on a separate plasmid or cell line) enables functional editing; dCas9 fusions support CRISPRi/a applications depending on the cassette architecture.

Best practices:

• Confirm absence/presence of Type IIS sites within the spacer for Golden Gate workflows.
• Sequence-verify junctions and the sgRNA region after cloning.
• Maintain plasmid in appropriate E. coli strains to minimize recombination if the vector contains repetitive elements.

Note: Specific restriction map and cloning sites for SKU P751464 are not provided; consult the CoA/Spec Sheet for exact features.

Target Specificity

Item-specific data required for target specificity (sgRNA spacer sequence, PAM context, species orthology, off-target analysis) are not provided for this SKU.

What can be inferred from the product name only:

• Putative target: U2AF1 gene. No sequence or exon/intron target site information is supplied.

General guidance (literature; not item-specific):

• Validate spacer identity by Sanger sequencing of the sgRNA insert.
• Use current design tools to predict on-target efficiency and off-target liabilities for the intended genome build.
• Confirm specificity empirically with amplicon NGS and orthogonal assays (e.g., GUIDE-seq, CHANGE-seq) when critical.

Conclusion:

• Specificity parameters for pLenti-U2AF1-sgRNA are not specified for this item; refer to the CoA/Spec Sheet and provide sequence-level details to your bioinformatics pipeline for in silico analysis.

자주 묻는 질문

How should this product be stored?

Store at ?80 °C. Ultra-low temperature storage is required to maintain the specified shelf life.

How is this product shipped?

This product ships frozen with dry ice and cold packs. Unpack on arrival and transfer it to the storage condition stated above; handle dry ice with insulated gloves in a ventilated area.

What documentation is provided?

Available product documentation, including Certificates of Analysis (COA), Safety Data Sheets (SDS), and specification sheets, is shown in the product document area. Document availability and access follow the current site policy.

Which species reactivity has this antibody been validated against?

Validated reactivity is listed on the PDP and on each lot's datasheet. We validate against the most commonly tested species for the target (pLenti-U2AF1-sgRNA); cross-reactivity in untested species may exist but is not guaranteed. Contact technical support for specific species inquiries.