pLenti-ABL2-sgRNA

Cat. No.: P743552

仕様

Product Name

pLenti-ABL2-sgRNA

Product Line

生物製品

保管と配送

保管条件

Store at -80°C

入荷

Dry ice packs + Cold packs

安定性と保管

Stored at -20 ℃, valid for at least 6 months;Stored at -80 ℃, valid for at least one year.

Application Protocols

Item-specific tested applications, recommended conditions, and positive controls: Not specified for this item; refer to CoA/Spec Sheet and SDS.

General recommendation

• Follow your institution’s standard operating procedures for cloning, transfection, and (if applicable) lentiviral packaging/transduction. Validate edits at the ABL2 locus using established genotyping workflows. For precise parameters (cell line, transfection reagent, antibiotic selection, MOI), consult the vector map and internal lab optimization data.

Biological Roles

Item-specific facts (from Product Data)

• Intended target: ABL2 (inferred from name). Exact spacer sequence and species specificity: Not specified for this item; refer to CoA/Spec Sheet.

Literature context on ABL2 biology (general; not item-specific claims)

• Gene/protein: ABL2 (also known as ARG) encodes a non‑receptor tyrosine kinase closely related to ABL1. It features SH3‑SH2‑kinase domains and C‑terminal actin‑binding regions.
• Cellular roles: ABL2 participates in actin cytoskeleton remodeling, cell adhesion, endocytosis, and signaling downstream of integrins and growth factor receptors. It modulates lamellipodia/filopodia dynamics and influences cell motility.
• Signaling interplay: ABL2 interfaces with pathways involving Src family kinases, CRK/CRKL adaptors, and Rho GTPase regulators, impacting phosphorylation cascades tied to morphology and migration.
• Genetic perturbation outcomes: Knockout or knockdown of ABL2 in model systems has been reported to alter cytoskeletal organization, migration/invasion phenotypes, and adhesion signaling; phenotypes may be cell‑type dependent. Use multiple guides and rescue experiments to ascribe specificity.
• Redundancy/compensation: Functional overlap with ABL1 can mask or modulate phenotypes; dual targeting strategies are sometimes employed in mechanistic studies. No clinical or therapeutic claims are made here; this information is provided to inform experimental design in research contexts.

Buffer Applications

Not typically applicable. This product is a plasmid DNA reagent, not a buffering agent. For routine handling, nuclease‑free water or low‑salt Tris‑EDTA buffers are commonly used (general guidance). Item‑specific buffer composition, if supplied, is not specified for this item; refer to CoA/Spec Sheet.

Green Alternatives

This product is a plasmid DNA tool; traditional solvent/energy metrics for “green chemistry” are not directly applicable. Nevertheless, process-level choices can reduce environmental and safety burdens.

General considerations (literature)

• Delivery method alternatives:
  • Non‑viral delivery (e.g., electroporation, lipid/polymer transfection) can avoid viral packaging, reducing BSL‑2 waste streams. Tradeoff: Usually lower stable integration efficiency compared to lentivirus.
  • RNP delivery (Cas9 protein + synthetic gRNA) eliminates DNA backbones and selection markers, shortens persistence, and can reduce off‑target window; however, it may require costly reagents and precise timing.
• Scale and waste minimization:
  • Use mini‑scale preps and low‑endotoxin kits that minimize guanidinium/phenol waste. Choose column chemistries with recyclable plastics when possible.
  • Pool viral preparations to avoid multiple small‑scale runs; filter and aliquot to minimize freeze–thaw losses.
• Energy use: Plan cloning steps to reduce overnight incubations; use room‑temperature Gibson mixes when appropriate to cut freezer/thermo‑cycler time. Select the approach that balances biosafety, efficiency, and waste minimization for your lab’s infrastructure.

Pharmaceutical Uses

Not applicable. This reagent is intended for research use only and is not formulated as a drug substance, excipient, or for any clinical manufacturing use. No pharmacopeial monograph or clinical formulation roles are provided for this item.

Physical Properties

Item-specific facts (from Product Data)

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Concentration, buffer, OD260/280, endotoxin: Not specified for this item; refer to CoA/Spec Sheet.

Computed/literature/general information (for context; not item-specific specs)

• Physical state: Aqueous solution or lyophilized DNA (varies by supplier); highly water‑soluble when in Tris/EDTA or nuclease‑free water.
• UV absorbance (literature): Nucleic acids A260 quantitation; 1 A260 unit ≈ 50 µg/mL for dsDNA. Typical A260/A280 ~1.8 for pure dsDNA.
• pH compatibility: DNA is stable near neutral pH (∼7–8); avoid acidic conditions that promote depurination.
• Temperature sensitivity: Repeated freeze–thaw cycles can shear/denature supercoiled DNA; store at ≤−20 °C for short term, −80 °C for long term (see item-specific storage below).
• Solubility: Readily soluble in aqueous buffers; insoluble in common organic solvents.
• Viscosity/handling: High‑copy plasmid preps can be viscous; pipet slowly to avoid shearing. Note: No boiling/melting point, density, refractive index, logP, or SMILES are applicable to macromolecular DNA.

Quality and Grades

Item-specific facts (from Product Data)

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

General information (literature/typical for plasmid DNA products)

• Typical QC for plasmids can include: restriction digest and/or complete sequencing of the insert/critical vector regions; A260/280 purity ratios; confirmation of supercoiled content; endotoxin testing for transfection suitability.
• Endotoxin considerations: Low‑endotoxin or “transfection‑grade” DNA improves performance in sensitive mammalian cells; if the grade is not stated, consider performing an endotoxin cleanup prior to demanding applications.
• Antibiotic marker and copy number: Influence yield and copy state in E. coli; verify on the vector map.
• Stabilizers/buffers: Plasmids may be supplied in nuclease‑free water or TE; EDTA chelates Mg2+ to protect from nucleases but may affect downstream enzymology at high concentrations. Item-specific buffer is not provided here.
• Trace contaminants: Residual salts, phenol, guanidinium, or RNA can inhibit enzymes and transfection; assess by test transfections or additional cleanup if necessary.

Reaction and Applications

Item-specific facts (from Product Data)

• Labeled use: pLenti-ABL2-sgRNA for research use only.
• Specific application validations: Not specified for this item; refer to CoA/Spec Sheet.

General applications (literature; not item-specific claims)

• CRISPR gene perturbation: Expression of an ABL2‑targeting sgRNA for use with co‑expressed or stably integrated Cas9 to induce double‑strand breaks at the ABL2 locus, enabling knockout via NHEJ or template‑directed edits via HDR when a donor is supplied.
• Lentiviral delivery workflows: pLenti backbones are commonly packaged into VSV‑G pseudotyped lentiviral particles using helper plasmids (gag/pol, rev, env) for efficient, stable delivery into dividing and non‑dividing mammalian cells. Perform under appropriate biosafety conditions.
• Functional genomics: Loss‑of‑function studies, pathway analysis, or synthetic lethality screening centered on ABL2 signaling networks.
• Derivatization/cloning: Spacer replacement to target alternate ABL2 sites or related kinases; subcloning sgRNA cassettes via Golden Gate or Gibson Assembly. Practical tips (literature)
• Validate on‑target activity by T7E1/TIDE/ICE or amplicon NGS. Use ≥2 independent sgRNAs to control for guide‑specific effects.
• Include non‑targeting and safe‑harbor (e.g., AAVS1) controls. Monitor potential off‑targets predicted in silico and by GUIDE‑seq/CIRCLE‑seq where critical.
• For lentiviral packaging, optimize MOI, polybrene (if used), and antibiotic selection based on the vector’s markers (confirm on the item’s map).

Reaction Conditions

Item-specific validated conditions are not provided for this product; refer to CoA/Spec Sheet. The following are general, literature-based guidelines for common workflows involving pLenti sgRNA plasmids (not item-specific claims):

• Cloning the spacer:
  • Golden Gate: Anneal phosphorylated oligos with 4‑bp overhangs; cycle with type IIS enzyme (e.g., BsmBI/BbsI) and T4 ligase. Typical cycling: 25–35 cycles between 37 °C/16 °C, then 50–60 °C enzyme kill.
  • Gibson Assembly: 15–60 min at 50 °C with 20–40 bp overlaps; transform chemically competent or electrocompetent E. coli.
• Plasmid propagation: Grow in appropriate antibiotic at 30–37 °C; avoid overgrowth to limit recombination. Use high‑copy strains if backbone permits.
• Transfection (plasmid delivery): Optimize DNA:reagent ratios per cell line; many adherent lines perform well near 0.5–2 µg DNA per well of a 6‑well plate. Validate viability and expression.
• Lentiviral packaging: Co‑transfect with gag/pol, rev, and VSV‑G plasmids in HEK293T/293FT cells. Harvest at 48–72 h; filter (0.45 µm). Determine titer by p24, qPCR, or antibiotic selection units. Perform under appropriate biosafety approvals.
• Editing assessment: Analyze 48–96 h post‑delivery; for stable lines, select for 3–7 days per vector marker and expand single‑cell clones for genotyping. Yields, titers, and efficiencies vary by backbone, insert, and cell type; empirical optimization is required.

Safety and Handling

Item-specific facts (from Product Data)

• GHS classification, signal word, pictograms, H‑statements: Not specified for this item; refer to SDS.
• Storage conditions: Store at −80 °C.
• Shipping: Dry ice packs + cold packs.
• Research use: For research use only.

General guidance (literature/typical for plasmid DNA; defer to SDS and institutional biosafety policies)

• Biosafety: Plasmid DNA encoding sgRNA in a lentiviral backbone is typically handled at BSL‑1 for cloning/propagation in non‑pathogenic E. coli. If used to produce lentiviral particles, packaging and transduction procedures generally require BSL‑2 or higher per institutional guidelines.
• PPE: Laboratory coat, safety glasses, and disposable gloves. Use nuclease‑free technique to avoid contamination/degradation.
• Incompatibilities: Avoid nucleases (DNase), strong acids/bases, and oxidizers. Do not autoclave the DNA solution; filter sterilize if needed.
• Hygiene: Work in a clean area; wipe surfaces with DNA‑degrading agents only when disposal is intended.
• First aid (overview): DNA solutions are generally of low hazard; in case of contact, rinse with water. Ingestion/inhalation unlikely under normal use. Seek medical attention if adverse symptoms occur. Always consult the SDS for authoritative hazard, spill, and disposal instructions.

Solvent Selection

This item is a plasmid DNA reagent; classical organic solvent selection is not typically applicable.

General guidance (literature)

• Medium: Use nuclease‑free water or low‑salt Tris‑EDTA (e.g., 10 mM Tris, 0.1–1 mM EDTA, pH 7.5–8.0) for long‑term stability. High ionic strength buffers can impact certain downstream enzymatic steps and transfection reagents.
• Compatibility: Avoid organic solvents; plasmid DNA is denatured/precipitated by alcohols at sufficient concentrations (e.g., ≥70% ethanol or isopropanol used intentionally for precipitation).
• Filtration/sterility: If sterile solutions are required, 0.22 µm filtration is preferred over heat sterilization. If you need guidance on a working buffer for a specific workflow (e.g., transfection, digestion, ligation), consult the protocol for that enzyme/system and the item’s CoA/Spec Sheet.

Storage and Reconstitution

Item-specific facts (from Product Data)

• Storage: Store at −80 °C.
• Shipping: Dry ice packs + cold packs.
• Reconstitution and buffer composition: Not specified for this item; refer to CoA/Spec Sheet.

General guidance (literature; not item-specific requirements)

• Aliquoting: Upon receipt, thaw on ice, gently mix, and aliquot to avoid repeated freeze–thaw cycles that can shear supercoiled DNA.
• Short‑term: For frequent use, store working aliquots at −20 °C; keep a master stock at −80 °C.
• Buffer choice: Nuclease‑free water for immediate transfection; TE (10 mM Tris, 0.1–1 mM EDTA, pH 7.5–8.0) for long‑term stability. Avoid high EDTA in enzyme reactions.
• Handling: Use low‑bind tubes and filtered tips. Do not vortex vigorously; flick or pipet gently.
• Stability: Protect from nucleases and repeated freeze–thaw. Verify integrity by agarose gel (supercoiled band) and purity by A260/A280 before critical experiments.

Structure and Identity

Item-specific facts (from Product Data)

• SKU: P743552
• Product name: pLenti-ABL2-sgRNA
• Type: Plasmid DNA reagent (lentiviral backbone implied by name) encoding an sgRNA targeting ABL2.
• Exact sequence, vector map, promoters, selection markers, and insert length: Not specified for this item; refer to CoA/Spec Sheet.

Computed/literature/general information (for context; not item-specific specs)

• Structural class: Circular double-stranded DNA plasmid in a lentiviral (pLenti) backbone used to express a CRISPR single‑guide RNA (sgRNA), typically under a small RNA polymerase III promoter (e.g., U6). Actual promoter and features must be confirmed on the item’s map.
• Functional groups: Nucleic acid polymer composed of deoxyribonucleotides; no small‑molecule SMILES/InChIKey applies.
• 2D/functional description in words: Closed-circular DNA backbone with essential vector elements (origin(s) of replication, selectable marker), containing a gRNA scaffold plus a gene‑specific 20‑nt targeting spacer for ABL2.
• Molecular formula, MW, SMILES, InChIKey, CAS, CID: Not applicable to plasmid DNA or not specified for this item; refer to CoA/Spec Sheet.

Synthetic Utility

This section is interpreted for molecular biology “construction” utility rather than small‑molecule synthesis.

General utility (literature; not item-specific claims)

• Modular cloning: pLenti sgRNA vectors typically accept spacer inserts via type IIS restriction (e.g., BsmBI/BbsI) or Gibson Assembly. This enables rapid retargeting to different ABL2 protospacers.
• Multi‑component builds: Duplex sgRNA cassettes or dual‑guide arrangements can be assembled for deletions or inversions across ABL2 loci. Barcodes or reporters can be inserted for lineage tracing.
• Vector engineering: Swapping promoters (e.g., U6 vs H1) or adding selection markers/fluorescent reporters supports selection and tracking post‑transduction. Ensure reading frame/terminators remain correct and avoid cryptic Pol III terminators in the spacer.
• Retrosynthetic perspective: Start from a verified backbone; plan insert assembly order to minimize repeated type IIS scars and to preserve PAM‑proximal sequence integrity. Sequence‑verify across junctions and the full sgRNA region.
• Downstream operations: The same backbone can be adapted for dCas9 fusions (CRISPRi/a) by replacing or co‑expressing the nuclease module; ensure appropriate scaffold and MS2/PP7 aptamers if using recruitment systems.

Target Specificity

Item-specific facts (from Product Data)

• Intended target: ABL2 (inferred from product name). Exact sgRNA spacer sequence, PAM context, and species: Not specified for this item; refer to CoA/Spec Sheet.

Notes

• Without the exact guide sequence and genome build, on‑target coordinates and predicted off‑targets cannot be provided here. Confirm specificity computationally (e.g., CRISPOR, Benchling) and experimentally (e.g., amplicon NGS) once the sequence is available.

よくある質問

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-ABL2-sgRNA); cross-reactivity in untested species may exist but is not guaranteed. Contact technical support for specific species inquiries.

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.