pLenti-CBLL1-sgRNA

Cat. No.: P744454

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

Application Protocols

The following are general, literature-based workflows for lentiviral sgRNA plasmids. Exact parameters (antibiotic selection, insert sequence, backbone features) are not specified for this item and must be confirmed from the CoA/Spec Sheet.

1. Bacterial amplification and QC

• Transform 10–50 ng plasmid into high‑efficiency chemically competent E. coli. Recover 1 h at 37°C in SOC.
• Plate on LB agar with the appropriate antibiotic (vector‑dependent). Incubate 12–16 h.
• Inoculate a single colony into 3–5 mL LB + antibiotic; expand to midiprep/maxiprep scale as needed.
• QC: restriction digest and Sanger sequencing of the sgRNA region.

2. Lentiviral production (BSL‑2)

• Seed HEK293T cells to 60–80% confluence (e.g., 10‑cm dish). Co‑transfect transfer vector + packaging + envelope plasmids using a suitable transfection reagent.
• Replace medium after 6–12 h. Harvest supernatant at 48 and 72 h; filter (0.45 µm). Optional concentration (PEG/ultracentrifugation).

3. Transduction and selection

• Transduce target cells at desired MOI (e.g., 0.3–1 for single‑copy integration). Include polycation (e.g., 4–8 µg/mL) if compatible. Spinoculation can improve efficiency.
• Begin selection 48–72 h post‑transduction according to the vector’s resistance marker (not specified). Maintain selection until uninfected controls are cleared.

4. Editing verification and assays

• Extract genomic DNA 3–7 days post‑selection; PCR the target locus; analyze indels (ICE/CRISPResso). Validate protein loss by immunoblot if applicable.

Always follow institutional biosafety, waste disposal, and approval requirements.

Biological Roles

The product name indicates an sgRNA targeting CBLL1; exact sequence and species specificity are not specified for this item and should be confirmed from the CoA/Spec Sheet. The following pertains to CBLL1 biology (literature/general):

• Gene: CBLL1 (also known as HAKAI) encodes an E3 ubiquitin ligase adaptor that recognizes phosphorylated E‑cadherin, promoting its ubiquitination and endocytosis. It participates in regulation of adherens junctions and epithelial cell–cell adhesion.
• Pathways and functions:
  • Modulates epithelial polarity and junction turnover; impacts cell motility and epithelial–mesenchymal transition programs in various models.
  • Interacts with growth factor signaling pathways (e.g., Src tyrosine kinase–dependent phosphorylation events) and may influence downstream signaling by controlling receptor/cadherin availability at the membrane.
  • Reported cytoplasmic and membrane‑proximal localization; contains a CBL‑like domain architecture adapted for ubiquitin pathway interactions.
• Experimental implications:
  • CRISPR-mediated perturbation of CBLL1 is commonly used to study cell adhesion dynamics, endocytosis of E‑cadherin, and ubiquitin signaling crosstalk.
  • Redundancy/compensation: Consider evaluating related ubiquitin adaptors and performing rescue with sgRNA‑resistant CBLL1 cDNA to confirm specificity.

No medical or clinical claims are made. Use this information to design controls and phenotypic assays relevant to junctional biology and ubiquitination.

Buffer Applications

This product is a plasmid DNA reagent; it is not a biochemical buffer component.

• Typical handling buffers (literature/general):
  • Storage/dilution: nuclease‑free water or TE buffer (10 mM Tris‑HCl, pH 7.5–8.0; 0.1–1 mM EDTA). Exact supplied buffer is not specified for this item; refer to CoA/Spec Sheet.
  • For enzymatic reactions (e.g., restriction digest, PCR, Gibson), use the enzyme‑supplier’s buffer systems and salt/Mg2+ conditions.
• Cell culture: For transfection, exchange into cell‑compatible solutions per reagent recommendations; for lentiviral supernatant, use sterile culture media and appropriate additives (e.g., polycations) following biosafety guidelines.

If you require a defined pH buffer system for chromatography, electrophoresis, or cell culture, select an appropriate buffering reagent (e.g., HEPES, PBS); this plasmid is not intended as a buffer.

Green Alternatives

Conventional “green chemistry” substitution is not directly applicable to a plasmid DNA reagent. However, there are greener strategic choices in genome engineering workflows (literature/general):

• Delivery strategy comparisons (environmental and safety perspectives):

  Option | Advantages | Considerations --- | --- | --- Lentiviral delivery (this vector class) | Stable genomic integration; broad tropism; useful for difficult cell types and pooled screens | Requires BSL‑2; generates biohazardous waste; packaging consumables and plastics footprint Non-viral transfection (electroporation, lipid) | No viral waste; simpler biosafety | Lower efficiency in some primary cells; often transient RNP delivery (Cas9+sgRNA complex) | No DNA integration; reduced off‑target window; minimal plasmid prep waste | Transient; requires protein sourcing; repeated dosing for stable effects

• Process improvements:

  • Use minibatch culture and high-yield plasmid prep kits optimized to reduce solvent/chaotropic salt usage; recycle plastics where validated.
  • Prefer endotoxin‑reduced preps only when necessary to avoid unnecessary processing steps.
  • Scale viral production to need; avoid overproduction to minimize waste.

These are general considerations; select the approach that balances biosafety, environmental impact, and experimental requirements.

Pharmaceutical Uses

Not applicable. This product is labeled for research use only and is not intended for diagnostic, therapeutic, or manufacturing use in humans or animals.

• Formulation/excipient status: Not pharmacopeial; no excipient role specified.
• Preclinical research context (literature/general): Lentiviral sgRNA constructs are widely used in discovery research, target validation, and mechanism studies in cell lines and primary cells. Any use beyond basic research would require purpose‑built, quality‑controlled materials and regulatory oversight, which is outside the scope of this product.

For any regulated applications, consult appropriate GMP suppliers and institutional/regulatory guidance.

Physical Properties

Item-specific physical/analytical values are not provided for this plasmid DNA construct.

• Appearance, concentration, solvent/buffer: Not specified for this item; refer to CoA/Spec Sheet.
• UV properties (literature/general for DNA):
  • Strong absorbance at 260 nm (A260 = 1.0 corresponds to ~50 µg/mL for dsDNA in 1 cm pathlength).
  • Typical purity ratios: A260/A280 ~1.8–2.0; A260/A230 >2.0 for clean DNA preps.
• pH, osmolality of supplied solution: Not specified for this item; refer to CoA/Spec Sheet. DNA is commonly supplied in nuclease‑free water or low-salt TE (10 mM Tris‑HCl, 1 mM EDTA; literature), but the exact buffer here is not specified.
• Thermal properties (literature/general for plasmid DNA):
  • DNA denaturation temperature depends on GC content and ionic strength; circular dsDNA is stable through routine lab handling on ice/ambient when nuclease-free.
• Solubility (literature/general):
  • Highly water soluble in nuclease‑free aqueous buffers; insoluble in organic solvents.
• Sterility/endotoxin: Not specified for this item; refer to CoA/Spec Sheet.
• DNase/RNase status: Not specified for this item; refer to CoA/Spec Sheet.

Quality and Grades

• Grade/Purity: Not specified for this item; refer to CoA/Spec Sheet.
• What to expect for plasmid reagents (literature/general):
  • Research-grade plasmid DNA is typically QC’d by restriction analysis and/or Sanger sequencing across critical regions (insert/gRNA scaffold). OD260/280, concentration, and supercoiled fraction may be reported by some suppliers.
  • Endotoxin level can be relevant for some transfection applications; absence or low levels may be offered as “endotoxin-reduced” grades. This is not specified for this item.
• Sequence/Insert Verification:
  • For sgRNA constructs, verify the 20‑nt spacer sequence and orientation; confirm absence of unwanted mutations in the scaffold and promoter regions via Sanger sequencing. Exact sequence information for this item is not specified.
• Stabilizers/format: Not specified for this item. Some plasmids are supplied in TE or water; stabilizers (e.g., EDTA) can chelate Mg2+ and reduce nuclease activity (literature). Check the CoA for the supplied buffer.
• Release documentation: For cGMP or clinical‑grade nucleic acids, extended testing (sterility, endotoxin, residuals) is required; this product is for research use only.
• Recommendation: On receipt, perform an incoming QC check suited to your workflow (miniprep from a single colony, restriction digest, and Sanger traces) before scale-up or viral packaging.

Reaction and Applications

This product supports genome engineering and functional genomics workflows rather than classical chemical reactions.

• Intended application (inferred from name; item specifics not provided): Expression of an sgRNA designed to target CBLL1 for CRISPR‑Cas9–mediated gene perturbation. Exact spacer sequence and vector features are not specified; verify via CoA/Spec Sheet.
• Typical uses (literature/general):
  • Gene knockout via SpCas9 nuclease (NHEJ-induced frameshifts).
  • CRISPRi/CRISPRa when sgRNA is paired with dCas9‑KRAB or dCas9‑VP64 systems.
  • Stable delivery using lentiviral particles for hard‑to‑transfect cell types and pooled screens.
• Practical notes:
  • Maintain high DNA integrity (supercoiled fraction) for efficient transfection and viral titer.
  • For lentiviral work, co‑transfect with appropriate packaging plasmids (e.g., second- or third‑generation systems; literature) and use BSL‑2 practices.
  • Validate on‑target editing by amplicon sequencing, T7E1, or ICE/CRISPResso analyses; confirm functional knockout at the protein level where applicable.
  • Assess off‑target risk in silico and empirically; design multiple sgRNAs per gene for robust phenotypes.
• Controls:
  • Non‑targeting sgRNA controls and sgRNAs to essential genes (e.g., POLR2A, RPA3) serve as positive/negative controls in screens (literature). Choose controls compatible with your Cas9 system.

Reaction Conditions

General, literature-based conditions relevant to workflows using lentiviral sgRNA plasmids. Item-specific parameters (e.g., antibiotic marker, exact backbone) are not specified and must be obtained from the CoA/Spec Sheet.

• Cloning of sgRNA spacer:
  • Oligo design: complementary 20‑nt spacer with appropriate overhangs for the vector’s Type IIS sites (e.g., BsmBI/BbsI; vector-dependent).
  • Annealing: 95°C 5 min; cool to room temperature over 30–60 min. Ligate into digested vector; transform high‑efficiency E. coli.
• Plasmid amplification:
  • Grow a single colony 12–16 h in selective LB (antibiotic per vector; not specified here). Use endo‑reduced maxiprep kits if transfecting sensitive cells.
• Transfection (packaging cells, e.g., HEK293T; literature):
  • Co‑transfect transfer vector with packaging plasmids using lipid or PEI; typical DNA mass ratios 4:3:1 (transfer:packaging:envelope) in 6‑well/10‑cm formats. Optimize per reagent.
  • Collect viral supernatant at 48 and 72 h; clarify by 0.45 µm filtration.
• Transduction of target cells:
  • Optional polycation (e.g., 4–8 µg/mL) and spinoculation to enhance MOI. For knockout screens, use MOI ~0.3–0.5; for single‑clone editing, MOI 0.3–1 to favor single integrants (literature).
  • Post‑transduction selection depends on vector marker (not specified).
• Editing validation: Harvest genomic DNA after 3–7 days; PCR amplicon and sequence (ICE/CRISPResso). Extend time for protein turnover as needed.

Follow institutional BSL‑2 practices for all lentiviral steps.

Safety and Handling

• GHS classification, pictograms, H‑statements, signal word: Not specified for this item; refer to SDS for authoritative safety information.
• Research Use: For research use only (per product data). Not for human or veterinary use, diagnostic, or therapeutic applications.
• Biosafety considerations (literature/general):
  • Plasmid DNA itself is non-infectious; however, because the backbone is described as “pLenti,” procedures that generate lentiviral particles (via co‑transfection with packaging plasmids) should be performed under BSL‑2/BSL‑2+ containment following institutional IBC approvals and NIH/WHO guidelines.
  • Employ physical containment, biological safety cabinets, appropriate waste decontamination (e.g., 10% bleach, autoclaving), and viral inactivation practices for any lentiviral work.
• PPE and handling:
  • Wear lab coat, gloves, and eye protection. Use nuclease‑free technique to prevent degradation and cross‑contamination.
  • Avoid aerosol generation during pipetting and sonication; wipe spills with DNA‑degrading and disinfecting solutions.
• Incompatibilities (general): Avoid nucleases and non-sterile solutions. Do not mix with strong oxidants or proteases; store away from heat and repeated freeze–thaw.
• First aid (general): In case of exposure to buffers, rinse with water. Seek medical attention if irritation persists. Report any accidental potential exposure to replication‑competent lentivirus (if produced) per institutional policy.
• Disposal: Decontaminate DNA solutions and consumables per local biosafety and hazardous waste regulations.

Solvent Selection

Not typically applicable. This is a plasmid DNA reagent intended for molecular biology workflows rather than organic synthesis or chromatographic separations.

• Working medium (literature/general): Use nuclease‑free water or TE buffer for dilution and storage; use sterile, buffered cell culture media for transfection/transduction applications.
• Miscibility: DNA is soluble in aqueous buffers; precipitates in the presence of alcohols and high salt (basis of ethanol/isopropanol precipitation).
• Practical tips:
  • Avoid organic solvents; they denature or precipitate DNA and can leave inhibitory residues for enzymatic steps.
  • For transfections, ensure buffer exchange to cell‑compatible solutions (e.g., endotoxin‑reduced water/TE) and follow reagent vendor guidance for complex formation.
• If your use case centers on organic reactions or solvent screening, this product is not suitable; consider small‑molecule reagents instead.

Storage and Reconstitution

• Storage conditions (item-specific): Store at −80°C (per product data). Minimize freeze–thaw cycles by aliquoting upon first thaw.
• Shipping (item-specific): Shipped on dry ice packs + cold packs.
• Format/buffer: Not specified for this item; refer to CoA/Spec Sheet. Commonly, plasmid DNA is supplied in nuclease‑free water or TE buffer (literature/general).
• Thawing and handling (general):
  • Thaw on ice. Briefly spin down contents before opening to avoid aerosolization.
  • Mix gently by pipetting; avoid vortexing to preserve supercoiled topology.
  • Use sterile, nuclease‑free tubes and tips. Keep on ice during setup.
• Aliquoting (general): Prepare single‑use aliquots sized for one transfection or packaging reaction to avoid repeated freeze–thaw.
• Short‑term storage (general): At 4°C for ≤24–48 h if actively using; return to −20°C/−80°C for longer periods. For maximal stability, retain at −80°C as specified for this item.
• Reconstitution (if lyophilized): Not specified for this item; refer to CoA/Spec Sheet. In general, reconstitute plasmid DNA in nuclease‑free water or TE to a convenient working concentration, then allow to dissolve on ice for 10–15 min before gentle mixing.
• Documentation: Record lot number, thaw/aliquot dates, and any buffer exchanges for traceability.

For research use only.

Structure and Identity

This product is a nucleic-acid research reagent, not a small-molecule chemical. It is described as a lentiviral sgRNA construct directed toward CBLL1.

• Item type (from name): pLenti-CBLL1-sgRNA, consistent with a lentiviral transfer plasmid encoding a single guide RNA (sgRNA) intended to target the CBLL1 gene. Exact sequence, vector map, promoter(s), selection marker(s), and cloning sites are not specified for this item; refer to CoA/Spec Sheet.
• CAS, CID, InChIKey, SMILES: Not applicable to plasmid DNA constructs.
• Molecular formula/MW: Not specified for this item; refer to CoA/Spec Sheet. (Plasmid MW depends on total base pairs; literature: ~660 Da per bp for double-stranded DNA.)
• Structural features (general/literature):
  • Circular double‑stranded DNA backbone typical of lentiviral “pLenti”-type transfer vectors.
  • An sgRNA expression cassette typically consists of a U6 or H1 RNA polymerase III promoter, a 20-nt spacer (gene‑targeting sequence), and a gRNA scaffold (literature, general). Exact cassette design is not specified for this item.
  • pLenti backbones generally include 5′ and 3′ LTRs, packaging signal (Ψ), and additional lentiviral elements (e.g., RRE, cPPT) enabling virus production when co-transfected with packaging plasmids (literature, general). Specific elements present here are not specified.
• 2D “structure in words”: circular plasmid DNA containing an sgRNA cassette nominally targeting CBLL1; additional functional modules typical of lentiviral vectors (promoters, selection marker, and lentiviral elements) may be present but are not item-specified.

Synthetic Utility

In the context of molecular biology (rather than organic synthesis), this plasmid serves as a versatile cloning and genetic perturbation scaffold.

• Functional modules (not item‑specific; literature/general for pLenti vectors): multiple cloning sites, promoters (e.g., U6/H1 for sgRNA; CMV/EF1α for transgenes), selection markers, and lentiviral elements. The exact modules present in this item are not specified; consult CoA/Spec Sheet.
• Typical transformations enabled:
  • Insertion or swapping of sgRNA spacers by oligo annealing/ligation or Golden Gate assembly (BbsI/BsmBI‑type cassettes common in CRISPR vectors).
  • Gibson/SLiCE/HiFi assembly for multi‑part cloning of cassettes or reporters.
  • Packaging into lentiviral particles for stable genomic integration in mammalian cells (requires separate packaging plasmids; literature).
• Retrosynthetic value for construct design:
  • Modular replacement of promoters, markers, and payloads enables rapid construction of derivative tools (e.g., multiplex sgRNA arrays, CRISPRa/i modules).
• Controls and verification:
  • Sequence verification of spacer and scaffold; restriction mapping to confirm backbone integrity; optional whole‑plasmid NGS for screens.

This framing emphasizes the reagent’s role as a synthetic biology vector rather than a substrate for chemical reactions.

Target Specificity

• Item-specific targeting details (spacer sequence, strand, species ortholog): Not specified for this item; refer to CoA/Spec Sheet.
• Based on the product name, the intended gene target is CBLL1. Verify the exact target site(s) and genome build before experiments.
• Off-target considerations (literature/general):
  • Use multiple independent sgRNAs per gene and employ in silico tools (e.g., CRISPOR, Benchling) to predict off‑targets.
  • Empirically assess editing specificity via targeted deep sequencing or GUIDE‑seq/CIRCLE‑seq where appropriate.
• Controls: Include non‑targeting and safe‑harbor‑targeting sgRNAs, and perform rescue with sgRNA‑resistant CBLL1 cDNA to confirm phenotype specificity.