pLenti-RNF186-sgRNA

Cat. No.: P750071

Specifications

Product Name

pLenti-RNF186-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

• Item-specific (from Product Data)
  • Tested applications and recommended conditions: Not specified for this item; refer to datasheet.
• General workflow for pLenti sgRNA use (not item-specific; example guidance only)
  1. Guide verification: sequence the sgRNA region to confirm spacer identity.
  2. Lentiviral packaging: co-transfect HEK293T with vector, packaging, and envelope plasmids using a lipid reagent in serum-free medium; swap to complete medium after 6–24 h.
  3. Harvest and clarify viral supernatant at 48–72 h; optionally concentrate (e.g., ultracentrifugation or PEG-based methods) following BSL-2 rules.
  4. Transduction: add viral supernatant to target cells, optionally with polybrene; incubate 12–24 h and replace medium.
  5. Selection: apply the appropriate antibiotic if the backbone encodes resistance (marker not specified for this item). Expand surviving cells.
  6. Assay editing: extract genomic DNA at 2–4 days; PCR-amplify RNF186 locus; analyze by T7E1, ICE/TIDE, or amplicon NGS. For CRISPRi/a, quantify RNF186 transcripts/protein by qPCR/Western.
  7. Clonal isolation (optional): dilute or sort single cells; expand and genotype.
• Notes
  • Optimize MOI, selection stringency, and assay timing per cell type. Follow institutional biosafety policies for viral work.

Biological Roles

• Item-specific (from Product Data)
  • Target specificity, organism, and guide sequence: Not specified for this item; refer to CoA/Spec Sheet.
• Literature/general context on RNF186 (not item-specific)
  • RNF186 encodes a RING-type E3 ubiquitin ligase reported to localize to endoplasmic reticulum membranes. E3 ligases confer substrate specificity in ubiquitin-mediated protein modification.
  • Putative roles include regulation of protein quality control and stress-responsive signaling via ubiquitination of specific substrates. Exact substrates and pathways can be cell-type dependent and remain under active study.
  • Genetic perturbation of RNF186 is used in research to dissect ER-associated degradation (ERAD) pathways, proteostasis, and downstream transcriptional programs.
• Reagent functional context (general)
  • An sgRNA directed to RNF186 enables targeted recruitment of Cas9/dCas9 systems to RNF186 coding or regulatory regions for knockout, repression, activation, or editing studies, facilitating genotype–phenotype analysis in cellular models.

Buffer Applications

• Not typically applicable
  • This product is a plasmid DNA construct and is not a buffering agent. It is commonly handled in nuclease-free water or TE buffer supplied by the manufacturer or prepared by the user.
• Practical note (general)
  • If reconstitution is required, use nuclease-free water or TE (10 mM Tris·HCl, 1 mM EDTA, pH 8.0). Exact buffer composition for this item is not specified.

Green Alternatives

• Context
  • This is a nucleic acid reagent; “green alternatives” relate to delivery methods and process choices rather than solvent substitution.
• Greener practice options (general, not item-specific)
  • Delivery approach: use non-viral delivery (electroporation or lipid nanoparticles) to avoid lentiviral packaging waste and BSL-2 requirements when feasible.
  • Media and plastics: minimize single-use plastics by consolidating preparations and using recyclable materials where permitted by biosafety rules.
  • Transfection reagents: choose less cytotoxic, biodegradable lipids or PEI alternatives when compatible with cell type and required efficiency.
  • Energy use: plan batch experiments to reduce freezer door openings and centrifuge runs; store working aliquots at −20 °C short term to limit −80 °C cycles if stability allows.
• Trade-offs
  • Non-viral methods may yield lower stable expression in difficult cell types; lentivirus provides high efficiency and long-term expression but requires additional containment and generates chemical/biological waste.

Pharmaceutical Uses

• Not applicable for this catalog item
  • This product is designated “For research use only.” It is not an excipient or an API and is not intended for diagnostic, therapeutic, or clinical manufacturing use.
• General information (not item-specific)
  • Plasmids are used in research-scale vector construction and cell-based assays. Any translation to GMP settings would require distinct GMP-grade materials, documentation, and validation not covered by this research-use product.

Physical Properties

• Item-specific (from Product Data)
  • Appearance: Not specified for this item; refer to CoA/Spec Sheet.
  • Concentration, buffer, A260/A280: Not specified for this item; refer to CoA/Spec Sheet.
• Literature/general values for plasmid DNA (not item-specific)
  • Form: circular double-stranded DNA (commonly supplied as an aqueous solution or as lyophilized DNA). Hydrated plasmids are water-miscible.
  • UV characteristics (literature): ε260 ≈ 20 (µg/mL)−1·cm−1 for dsDNA; A260/A280 ~1.8–2.0 for pure DNA.
  • Solubility: plasmid DNA is soluble in aqueous buffers (e.g., TE, nuclease-free water); insoluble in nonpolar organic solvents.
  • Stability: highly dependent on nuclease-free handling, pH ~7.5–8.5, and low temperature storage (−20 to −80 °C). Repeated freeze–thaw can cause nicking.
  • Physical state: supercoiled topology predominates in high-quality prep; nicked/open circular or linear forms may be present after stress.
• Notes
  • Boiling point, melting point, density, logP, refractive index are not typically applicable to macromolecular DNA solutions and are not specified for this item.

Quality and Grades

• Item-specific (from Product Data)
  • Grade/purity: Not specified for this item; refer to CoA/Spec Sheet.
• General guidance on quality for plasmid DNA (not item-specific)
  • Research-use plasmids are commonly qualified by A260/A280 ratio (~1.8–2.0), supercoiled content (e.g., agarose gel densitometry), and endotoxin level (for cell culture applications). Low-endotoxin or transfection-grade DNA is preferred for sensitive primary cells and lentiviral packaging.
  • Sequence identity and integrity are typically confirmed by restriction digest and/or NGS/Sanger sequencing around the sgRNA cassette and vector junctions. For CRISPR work, verification of the 20-nt spacer sequence and PAM context is essential.
  • Antibiotic resistance marker accuracy, backbone length, and selectable markers should match the vector map; these details are not specified for this item.
  • Stabilizers: Plasmid DNA is often provided in TE (10 mM Tris·HCl, 1 mM EDTA, pH 8.0) to chelate Mg2+ and inhibit DNases; exact buffer for this item is not specified.

Reaction and Applications

• Item-specific (from Product Data)
  • Manufacturer applications: Not specified in the listing; refer to product datasheet. Product name indicates an sgRNA plasmid in a lentiviral backbone targeting RNF186.
• General applications for pLenti sgRNA constructs (not item-specific)
  • CRISPR workflows: expression of a single guide RNA under a Pol III promoter to direct Cas9 or Cas9 derivatives to RNF186 genomic loci.
  • Use cases:
    • Gene knockout (SpCas9 nuclease creating indels via NHEJ).
    • Transcriptional repression (CRISPRi with dCas9–KRAB) or activation (CRISPRa with dCas9–VP64/p300) at RNF186 regulatory regions.
    • Base/prime editing when paired with compatible editors and appropriately designed guides.
  • Lentiviral delivery enables stable integration or long-term expression across dividing/non-dividing cells; typical in hard-to-transfect lines.
• Practical tips (general)
  • Guide validation: in silico off-target prediction and on-target efficiency scoring, then empirical testing via T7E1, ICE/TIDE, or amplicon NGS.
  • Packaging: co-transfect with 2nd/3rd generation helper plasmids in HEK293T; harvest viral supernatants at 48–72 h; titer by p24, qPCR, or functional assays.
  • Transduction: use polybrene (if compatible) and spinoculation as needed; select with the vector’s antibiotic marker (if present; not specified for this item).
  • Confirm editing at RNF186 by sequencing and/or protein-level assays; isolate clonal lines when precise genotypes are required.

Reaction Conditions

• Not applicable to chemical reaction conditions
  • Traditional parameters such as temperature/solvent/catalyst for chemical synthesis do not apply to a plasmid DNA reagent.
• General molecular biology conditions (not item-specific)
  • Transfection for packaging: commonly perform in HEK293T cells at 37 °C, 5% CO2, using lipid-based reagents in serum-free medium; replace with complete medium after 4–24 h.
  • Viral harvest: collect supernatants at ~48 and ~72 h post-transfection; clarify by low-speed spin and 0.45 µm filtration.
  • Transduction: expose target cells to viral supernatant with or without polybrene (2–8 µg/mL) for 12–24 h; selection window and antibiotic depend on vector marker (not specified for this item).
  • Editing window: assess indels 48–96 h post-transduction for nuclease applications; allow longer for clonal isolation. Parameters are general guidance and must be optimized per cell type.

Safety and Handling

• Item-specific (from Product Data)
  • GHS/Signal word/H-statements/Pictograms: Not specified for this item; refer to SDS.
  • Storage: Store at −80 °C.
  • Shipping: Shipped on dry ice packs + cold packs.
• General laboratory biosafety (not item-specific)
  • Handle plasmid DNA and lentiviral vector backbones using BSL-1 practices; any generation of lentiviral particles requires BSL-2 containment and institutional approvals. Follow institutional biosafety committee (IBC) policies.
  • Avoid nuclease contamination: wear gloves, use nuclease-free tips/tubes, and dedicated aliquots.
  • PPE: lab coat, gloves, and eye protection. Avoid aerosol generation when pipetting.
  • Incompatibilities: DNases (from skin, non-sterile plastics), extreme pH (<6 or >9), prolonged room-temperature exposure, repeated freeze–thaw cycles.
  • First aid (general): if contact with eyes/skin occurs, rinse with water. If inhaled as aerosol or ingested, seek medical attention per SDS guidance.
  • Decontamination: 10% bleach effectively inactivates residual nucleic acids and lentiviral particles; follow local waste disposal regulations for GMO materials.
• Authoritative source
  • Always consult the product-specific SDS and your institution’s biosafety manual for definitive hazard classification and handling procedures.

Solvent Selection

• Applicability
  • This product is a plasmid DNA construct and is not used as a reaction solvent. Traditional solvent selection criteria (polarity, dielectric constant) do not apply.
• Practical notes (general, not item-specific)
  • Resuspension/handling: use nuclease-free water or TE buffer (pH 7.5–8.5). Avoid divalent cations unless chelated by EDTA.
  • For transfection, dilute in sterile, serum-free media or the specific buffer recommended for your transfection reagent (e.g., Opti-MEM for some lipids). Follow reagent manufacturer guidance.
  • Avoid organic solvents; they can denature or precipitate DNA and are incompatible with mammalian cells.

Storage and Reconstitution

• Item-specific (from Product Data)
  • Storage conditions: Store at −80 °C.
  • Shipped in: Dry ice packs + Cold packs.
• General guidance for plasmid DNA (not item-specific)
  • Upon receipt: keep frozen; minimize freeze–thaw cycles by preparing single-use aliquots on ice.
  • Short-term: store working aliquots at −20 °C; long-term at −80 °C for maximal stability.
  • Reconstitution: if supplied lyophilized, reconstitute with nuclease-free water or TE buffer to the desired concentration; mix gently without vortexing to preserve supercoiling.
  • Handling: use nuclease-free, low-retention tubes and tips. Avoid repeated freeze–thaw; thaw on ice and keep cold during setup.
  • Stability: maintain pH 7.5–8.5; EDTA (0.1–1 mM) helps chelate Mg2+ and protect against DNases. Do not store in buffers containing Ca2+/Mg2+ without EDTA.
  • Documentation: consult the product’s CoA/Spec Sheet for any item-specific buffer composition, concentration, and recommended storage durations (not specified here).

Structure and Identity

• Item-specific (from Product Data)
  • SKU: P750071
  • Product name: pLenti-RNF186-sgRNA
  • Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
  • Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.
  • CAS, CID, InChIKey, SMILES: Not applicable/not specified for plasmid DNA constructs.
• Literature/general description (not item-specific)
  • pLenti-sgRNA reagents are typically circular double-stranded DNA plasmids engineered for lentiviral delivery of a single guide RNA (sgRNA) under a Pol III promoter (commonly U6 or H1).
  • The construct name suggests an sgRNA designed to target the RNF186 gene (ring finger protein 186). Exact spacer sequence, PAM context, and scaffold are not specified for this item.
  • Common structural features in pLenti sgRNA backbones include: 5′ LTR, packaging signal (Ψ), Rev response element (RRE), central polypurine tract (cPPT), U6 promoter → sgRNA scaffold cassette, and 3′ SIN-LTR; selectable markers (e.g., puromycin resistance) and reporter cassettes may be present depending on backbone, but are not specified here.
  • 2D conceptual structure: circular plasmid map with distinct functional modules (promoters, ORFs, selection markers, LTRs) arranged along the backbone; the sgRNA cassette comprises a 20-nt spacer upstream of a conserved tracrRNA-derived scaffold.

Synthetic Utility

• Not applicable to classical chemical synthesis
  • As a biological plasmid reagent, this product is not used as a small-molecule building block or catalyst.
• Molecular biology utility (general, not item-specific)
  • Serves as a vector for expressing an RNF186-targeting sgRNA to recruit CRISPR effectors, enabling genome engineering workflows, pathway interrogation, and functional genomics screens.

Target Specificity

• Item-specific (from Product Data)
  • Target, guide sequence, PAM context, and species specificity: Not specified for this item; refer to CoA/Spec Sheet.
• General notes (not item-specific)
  • An “RNF186-sgRNA” designation implies intended targeting of the RNF186 gene. Actual on-target cut site, strand orientation, and off-target profile depend on the exact 20-nt spacer, Cas variant used, and genome build.
  • Verification recommended: confirm target engagement by amplicon sequencing at the intended RNF186 locus and screen predicted off-target sites using in silico tools and targeted assays.