pLenti-CBLL1-sgRNA

Cat. No.: P744453

Specifications

Product Name

pLenti-CBLL1-sgRNA

Product Line

Biologische Produkte

Lagerung und Versand

Storage

Store at -80°C

Verschickt in

Dry ice packs + Cold packs

Stabilität und Lagerung

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

Application Protocols

Tested/validated applications, recommended dilutions, and step-by-step protocols are Not specified for this item; refer to the product CoA/Spec Sheet and SDS.

Note

• Because this is a research-use plasmid, laboratories typically adapt standard CRISPR cloning, transfection, and lentiviral transduction protocols to their cell models. However, no application has been validated or warranted by the supplier for this specific SKU in the provided data.

Biological Roles

Context on the target gene (literature; not item-specific to the construct)

• CBLL1 (Cbl proto-oncogene like 1), also known as HAKAI, encodes an E3 ubiquitin ligase containing a RING finger domain. It recognizes phosphorylated E-cadherin and promotes its ubiquitination and endocytosis, thereby modulating adherens junction turnover and epithelial cell–cell adhesion dynamics.
• Pathways: Implicated in regulation of cell adhesion, polarity, and signaling through interactions impacting cadherin complexes and Src kinase pathways (literature). It may influence epithelial–mesenchymal transitions in developmental and cellular models.
• Cellular localization: Predominantly cytoplasmic with membrane-associated activities linked to adherens junctions (literature).

Relevance to experimental design

• Knockout or knockdown of CBLL1 can be used to interrogate pathways controlling cadherin stability, ubiquitin-mediated trafficking, and cytoskeletal organization in epithelial cell models.
• Overexpression/perturbation studies can reveal compensatory mechanisms in ubiquitin ligase networks.

Notes

• The above information provides biological background to guide assay selection and phenotypic readouts. It does not imply any medical or clinical application.
• Exact spacer sequence and any on-/off-target predictions for this specific item are not provided here; consult CoA/Spec Sheet if available.

Buffer Applications

Applicability

• As a plasmid DNA reagent, classic buffering concepts (acid/base conjugate pairs) are less relevant than nuclease-inhibitory and DNA-stabilizing buffers.

General guidance (literature; not item-specific)

• TE buffer: 10 mM Tris-Cl, pH 7.5–8.5 + 0.1–1 mM EDTA. Offers improved resistance to nuclease degradation during storage by chelating Mg2+/Mn2+.
• Tris buffer (no EDTA): 10 mM Tris-Cl pH 7.5–8.0 for applications sensitive to chelators (e.g., Mg2+-dependent PCR, certain transfections). Add salts only as needed.
• Nuclease-free water: Suitable for short-term storage and many enzymatic reactions; less protective long-term.

Practical tips

• Adjust pH at room temperature; Tris pH shifts with temperature (~0.03 pH/°C; literature).
• Filter-sterilize (0.22 µm) and use DNase/RNase-free plastics. Avoid repeated freeze–thaw by aliquoting.

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

Green Alternatives

While plasmid DNA itself is relatively benign, upstream and downstream workflows can be optimized for greener, safer practices (general guidance; not item-specific).

• DNA purification

  • Prefer silica membrane or magnetic-bead kits with fewer hazardous organic solvents instead of traditional phenol–chloroform extractions.
  • Choose suppliers offering solvent-minimized or recyclable packaging.

• Electrophoresis and staining

  • Replace ethidium bromide with safer DNA stains (e.g., SYBR Safe, GelRed) and use blue-light transilluminators to avoid UV exposure.

• Delivery methods

  • Where appropriate, consider Cas9 RNP delivery to avoid viral packaging waste streams; however, balance against the need for stable sgRNA expression that lentiviral vectors provide.

• Waste management

  • Segregate biohazard and chemical waste. Neutralize bleach before drain disposal per local regulations; minimize single-use plastics by adopting reusable gel trays and reservoirs.

Comparison (general)

• Phenol–chloroform vs. spin-column: Spin-column reduces hazardous waste at a small cost increase and potential silica waste.
• UV vs. blue light for gel visualization: Blue light reduces DNA damage and user UV exposure, improving cloning success.

Note: These are general green practice suggestions; they do not modify the product’s specifications or performance claims.

Pharmaceutical Uses

This product is designated for research use only and is not manufactured to GMP standards. It is not an excipient, drug substance, or diagnostic material.

Potential roles in pharmaceutical R&D workflows (general; not item-specific)

• Target validation: Gene perturbation of CBLL1 in cell lines to study pathway relevance during early discovery.
• Assay development: Creation of isogenic knockout/knockdown lines to build robust screening cascades and mechanistic assays.
• Process development research: Benchmarking transfection/transduction methods and selection systems; not for clinical manufacturing.

Regulatory status

• No pharmacopeial monograph applies to this research plasmid. Any use in regulated environments would require separate qualification and/or custom GMP production.

Item-specific pharmacopeial or GMP details: Not specified for this item; refer to CoA/Spec Sheet.

Physical Properties

Item-specific physical 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.
• Melting/boiling points, density, refractive index: Not applicable to plasmid DNA.

General/literature characteristics for plasmid DNA solutions (informational; not item-specific)

• Molecular form: Circular double-stranded DNA, predominantly supercoiled under standard prep conditions.
• Size: Common pLenti transfer vectors are ~8–12 kb, but exact size varies by backbone and inserts.
• Solubility: Readily soluble in nuclease-free aqueous buffers (e.g., TE, Tris, or nuclease-free water). Insoluble in most organic solvents.
• UV absorbance: DNA shows a peak at ~260 nm; A260 of 1.0 corresponds to ~50 µg/mL for dsDNA (literature).
• pH stability: Most plasmids are stable near neutral pH; hydrolysis and depurination increase <pH 6 or >pH 9 (literature).
• Thermal behavior: Denaturation/strand separation monitored by hyperchromicity near 80–95°C depending on GC content and ionic strength (literature). Routine storage is frozen to minimize nuclease activity and depurination.

Practical notes

• Avoid repeated freeze–thaw cycles to maintain supercoiling and integrity.
• Use low-EDTA buffers if downstream enzymology or cell transfection is sensitive to chelators.

For any definitive numeric specifications (concentration, solvent/buffer, endotoxin, residual solvents): Not specified for this item; refer to CoA/Spec Sheet.

Quality and Grades

Item-specific quality details

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

How to interpret typical quality indicators for plasmid DNA (general guidance; not item-specific)

• Identity confirmation: Plasmid map concordance by diagnostic restriction digest and/or full insert Sanger sequencing to verify the sgRNA spacer, scaffold, and junctions.
• Purity assessments: A260/A280 ratio ~1.8–2.0 (dsDNA, literature) and intact supercoiled banding on agarose gel. Absence of RNA (A260/A230), protein, phenol, and salts.
• Endotoxin level: For cell culture and transfection, low endotoxin preparations (<0.1 EU/µg DNA; literature best-practice) improve performance. Exact endotoxin spec is not provided for this item.
• Topology: High supercoiled content generally correlates with higher transfection efficiency; nicked/linear content should be minimized.
• Sterility: Filtered through 0.22 µm and prepared in nuclease-free conditions to reduce contamination.

What the grade implies

• If designated as “research grade” (typical for plasmids), it indicates suitability for laboratory research. It is not manufactured to GMP standards and is not for diagnostic or therapeutic use.

Documentation

• For definitive specifications (antibiotic selection marker, backbone version, insert sequence, concentration, endotoxin, QC methods): Not specified for this item; refer to CoA/Spec Sheet.

Reaction and Applications

Scope (general/literature context for pLenti sgRNA constructs; not item-specific)

• Genome editing: Provides a single-guide RNA (sgRNA) cassette targeting the CBLL1 gene for CRISPR-Cas9-mediated DNA cleavage and knockout/perturbation studies.
• Lentiviral delivery: pLenti backbones enable packaging into VSV-G–pseudotyped lentiviral particles for efficient transduction of dividing and non-dividing mammalian cells, supporting stable integration and long-term expression of the sgRNA.
• Functional genomics: Use in pooled or arrayed loss-of-function screens focusing on pathways where CBLL1 is implicated (e.g., ubiquitination of cell adhesion proteins; literature background).
• Complementary applications: Knock-in/repair experiments when co-delivered with donor templates; CRISPRi/CRISPRa if the cassette is adapted to dead-Cas9 fusions (dCas9-KRAB/VP64) and appropriate promoters (general).

Practical tips (general best-practices)

• Spacer validation: In silico on-target/off-target analysis against the host genome; verify presence and orientation by Sanger sequencing.
• Activity controls: Include non-targeting sgRNA and known positive-control sgRNA to benchmark editing efficiency.
• Delivery choice: Plasmid transfection vs. lentiviral transduction. Viral delivery requires BSL-2 practices and appropriate packaging plasmids.
• Readouts: TIDE/ICE analysis, amplicon NGS, or functional phenotyping; confirm allelic edits and potential indels at predicted off-targets.

Item-specific application notes from catalog: Not specified for this item; refer to CoA/Spec Sheet.

Reaction Conditions

Because this is a plasmid reagent, “reaction conditions” map to cloning, transfection, and viral packaging steps. The following are general literature practices and are not item-specific.

• Cloning/editing of the sgRNA spacer

  • Golden Gate: Annealed oligos with 4-nt overhangs ligated using BsmBI/BbsI in one-pot cycles (37°C digestion/16°C ligation cycling). Verify by Sanger sequencing.

• Transfection into mammalian cells

  • DNA amounts: Commonly 0.5–2 µg per well (6-well format), optimized per cell line and reagent. Serum and antibiotics may affect efficiency.
  • Post-transfection: Replace medium after 6–24 h depending on reagent guidance. Assess editing after 48–96 h (TIDE/ICE) or after selection.

• Lentiviral packaging (BSL-2; institutional approvals required)

  • Producer cells: HEK293T or similar; co-transfect with packaging plasmids (e.g., Gag/Pol and VSV-G). Harvest supernatant at 48–72 h. Filter (0.45 µm) and optionally concentrate.
  • Transduction: Add polybrene (e.g., 4–8 µg/mL) as compatible; select with appropriate antibiotic if the transfer vector encodes one. Determine MOI empirically.

• Downstream validation

  • Genotyping: PCR across target site and Sanger/NGS; functional assays tailored to CBLL1 biology (e.g., E-cadherin surface levels; literature-derived concept).

Exact conditions, plasmid maps, packaging mixes, and antibiotic selections for this item: Not specified for this item; refer to CoA/Spec Sheet.

Safety and Handling

Authoritative safety guidance must be taken from the product-specific SDS.

• GHS classification and labeling (from Product Data)

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

• Biosafety considerations (general for lentiviral plasmids; not item-specific)

  • The item is a plasmid DNA; it is not infectious by itself. When used to generate lentiviral particles via packaging in producer cells, treat all procedures as BSL-2 with appropriate institutional approvals and training.
  • Prevent dissemination of recombinant lentivirus: use biological safety cabinets, sealed rotors, and validated chemical disinfectants (e.g., freshly prepared 10% bleach followed by 70% ethanol) for spills.

• Handling and PPE (good laboratory practice)

  • Wear lab coat, safety glasses, and disposable nitrile gloves. Employ nuclease-free technique to avoid degradation and potential contamination.
  • Avoid aerosol generation; cap tubes promptly. Decontaminate work surfaces before and after use.

• Incompatibilities and stability (general)

  • Avoid nucleases (DNase), strong acids/bases, and prolonged elevated temperatures. Do not expose to UV light without shielding.

• First-aid overview (general)

  • Skin/eye contact with buffers: rinse with water. If ingested or inhaled as aerosolized buffer, move to fresh air and seek medical attention as needed. Always follow institutional emergency procedures.

• Storage/shipping (from Product Data)

  • Storage: Store at -80°C.
  • Shipped: On dry ice packs + cold packs.

Solvent Selection

Applicability

• This is a nucleic acid reagent typically supplied in an aqueous buffer. Organic solvent selection is not relevant. Focus on nuclease-free aqueous media.

General guidance (literature; not item-specific)

• Preferred solvents/buffers: Nuclease-free water, 10 mM Tris-Cl pH 7.5–8.5, or TE buffer (10 mM Tris, 0.1–1 mM EDTA). EDTA chelates divalent cations to inhibit DNases; however, EDTA may inhibit enzymatic reactions and some transfections.
• Ionic strength: Inclusion of 50–150 mM NaCl can increase DNA stability in some contexts but may impact downstream applications.
• Avoid: Alcohols, strong acids/bases, and organic solvents that precipitate or denature DNA.

Selection tips

• For cloning/enzymatic reactions: Use low-salt Tris or nuclease-free water; minimize EDTA.
• For cell transfection: Many reagents tolerate Tris or water; some perform best in water without EDTA/salt.
• For long-term storage: TE buffer provides better stability against nucleases; aliquot to avoid freeze–thaw.

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

Storage and Reconstitution

Item-specific storage and shipping (from Product Data)

• Storage Conditions: Store at -80°C.
• Shipped In: Dry ice packs + Cold packs.

General guidance for handling plasmid DNA (not item-specific)

• Upon receipt: Keep frozen. Minimize time at ambient temperature. Record lot information.
• Aliquoting: Thaw once on ice, mix gently (avoid vortexing to preserve supercoiling), spin briefly, and aliquot into nuclease-free, low-bind tubes to avoid repeated freeze–thaw cycles.
• Working storage: -20°C for short-to-medium term; -80°C for long-term. Avoid frost-free freezers. Protect from light.
• Thawing: On ice. Mix by gentle flick or slow pipetting. Do not heat.
• Buffer considerations: TE buffer enhances stability for storage; nuclease-free water may be preferred for immediate enzymatic reactions. Exact buffer for this item is not specified.
• Sterility: Use DNase/RNase-free tips and tubes. Wipe down work area with DNA decontaminant.

Reconstitution

• If supplied lyophilized (not specified), reconstitute in nuclease-free water or low-EDTA Tris to the desired concentration, allow to sit on ice 10–15 min, and mix gently. For any item-specific reconstitution instructions: Not specified for this item; refer to CoA/Spec Sheet.

Research Use

• For research use only (from Product Data).

Structure and Identity

Brief description: pLenti-CBLL1-sgRNA is a lentiviral CRISPR guide RNA construct intended for research use in manipulating the CBLL1 locus.

• Item-specific identifiers (from Product Data)

  • SKU: P744453
  • Product Name: pLenti-CBLL1-sgRNA
  • CAS: Not applicable for plasmid DNA
  • 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 large DNA constructs; not specified for this item.

• Structural nature (general/literature description for pLenti sgRNA vectors; not item-specific)

  • Macromolecule type: Circular double-stranded DNA plasmid, typically several kilobases in length (literature: ~8–12 kb for common pLenti backbones).
  • Functional cassettes commonly present: 5′/3′ LTRs (self-inactivating in modern designs), ψ packaging signal, bacterial origin of replication and antibiotic resistance marker, and a U6 or H1 promoter-driven sgRNA scaffold containing a 20 nt spacer specific to CBLL1.
  • The “CBLL1-sgRNA” denotes the spacer sequence is designed to target the CBLL1 gene (also known as HAKAI); exact spacer sequence and PAM context are not specified for this item.

• 2D/sequence-level description (general)

  • Topology: supercoiled plasmid in propagation; linearized upon specific restriction digestion.
  • Key features are arranged as discrete modules: bacterial selection/replication region, lentiviral transfer region, and a Pol III promoter followed by sgRNA scaffold and terminator.

Notes

• Exact map, base pair length, antibiotic marker, promoter choices, and sgRNA spacer sequence: Not specified for this item; refer to CoA/Spec Sheet.

Synthetic Utility

Interpretation for a molecular-biology construct (general; not item-specific)

• Functional modules enabling modification

  • sgRNA spacer region: Can be re-targeted to alternative CBLL1 loci or other genes via oligo cloning (e.g., BsmBI/BbsI Golden Gate) while retaining the same scaffold.
  • Promoter elements: Pol III promoters (U6/H1) commonly drive sgRNA expression; swapping promoters can modulate expression across cell types.
  • Selection markers: Bacterial (e.g., AmpR) and mammalian (e.g., puromycin) markers, if present, permit propagation and selection; exact markers for this item are not specified.

• Typical transformations

  • Golden Gate cloning to insert new spacers or multiplex sgRNAs (tandem cassettes).
  • Gibson Assembly or restriction–ligation to introduce additional expression cassettes (e.g., reporters, selectable markers).
  • Packaging into lentiviral particles for stable genomic integration in target cells.

• Retrosynthetic value

  • The modular plasmid map supports rapid reconfiguration: backbone + promoter + sgRNA scaffold + spacer. This architecture facilitates iterative design–build–test cycles in CRISPR workflows.

Item-specific vector map, restriction sites, and markers: Not specified for this item; refer to CoA/Spec Sheet.

Target Specificity

Only item-specific details are reported here.

• Intended gene target: CBLL1 (by product name), but the exact spacer sequence, PAM context, transcript isoform targeting, predicted off-targets, and validated on-target activity are Not specified for this item; refer to CoA/Spec Sheet.
• Antigen/epitope, species reactivity, clone, isotype: Not applicable; this is not an antibody reagent despite catalog category labeling.

For definitive targeting information (guide sequence, genomic coordinates, strand orientation, and screening data): Refer to the product CoA/Spec Sheet.