pLenti-ARG2-sgRNA

Cat. No.: P743869

Specifications

Product Name

pLenti-ARG2-sgRNA

Product Line

biological_products

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Store at -80°C

Enviado en

Dry ice packs + Cold packs

Estabilidad y almacenamiento

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

Application Protocols

No vendor-validated application protocols are provided for this specific item. The following outlines common workflows for sgRNA plasmids (general, literature):

1. Resuspension and aliquoting

• Upon receipt on dry ice/cold packs, keep frozen. Thaw on ice. If provided lyophilized, resuspend in nuclease-free water or TE to 50–500 ng/µL. Vortex gently or pipette to dissolve. Spin briefly and aliquot into DNase/RNase-free tubes.

2. Bacterial amplification and QC

• Transform into competent E. coli; select on the appropriate antibiotic (not specified). Miniprep colonies and verify the sgRNA insert by Sanger sequencing using vector-specific primers. Choose the correct clone for scale-up and midiprep/maxiprep. Assess A260/280 and run an agarose gel to confirm supercoiled topology.

3. Transfection or lentiviral packaging

• For plasmid transfection: Optimize DNA amount and reagent ratios for your cell line. For lentiviral production: Co-transfect with packaging/envelope plasmids, collect supernatant at 48–72 h, filter, and transduce target cells under BSL‑2 conditions. Apply selection encoded by the vector if applicable (marker not specified).

4. Editing validation

• Extract genomic DNA 48–96 h post-delivery. Amplify the ARG2 target region and quantify editing by ICE/TIDE or amplicon NGS. Confirm phenotype with orthogonal assays and include non-targeting/positive control guides.

Note: Exact buffer, antibiotic marker, sequence, and titration recommendations are not specified for this item; refer to CoA/Spec Sheet.

Biological Roles

This plasmid encodes an sgRNA designed to target the human ARG2 gene; it is a tool to perturb biological pathways for research.

ARG2 biology (literature/general):

• Gene/protein: Arginase 2 (ARG2) is a mitochondrial enzyme that hydrolyzes L‑arginine to L‑ornithine and urea. It is distinct from cytosolic ARG1 and is expressed in various tissues.
• Pathway context: Competes with nitric oxide synthase for L‑arginine, thereby influencing nitrogen/urea metabolism and cellular polyamine/ornithine pools. Alterations can affect redox balance and immune/metabolic signaling.
• Cellular localization: Mitochondrial matrix targeting via N‑terminal sequence (literature/general).

Research utility of ARG2 targeting (literature/general):

• Functional genomics: Knockout/knockdown enables dissection of arginine metabolism, urea cycle contributions outside the liver, and metabolic remodeling.
• Systems biology: Studying flux changes in arginine–ornithine–polyamine pathways using isotope tracing after ARG2 perturbation.
• Synthetic biology: Integration with inducible Cas systems or multiplex sgRNAs to map gene–gene interactions within nitrogen metabolism.

Note: The exact guide sequence and nuclease compatibility for this item are not specified and determine on-target cut site(s) and off-target risk; consult the CoA/Spec Sheet/sequence file. All uses are for research purposes only.

Buffer Applications

While conventional buffer systems (e.g., pH buffers for chromatography) are not the primary focus for a plasmid DNA product, appropriate aqueous buffers are essential for stability and performance.

• Resuspension buffers (general, literature):

  • Nuclease-free water: Suitable for immediate downstream enzymology (PCR, cloning). Store cold and use promptly.
  • TE buffer (10 mM Tris-HCl, pH 7.5–8.0; 0.1–1 mM EDTA): Enhances stability during storage by chelating Mg2+ required by nucleases.
  • Low-EDTA Tris buffer: Preferred when immediate enzymatic reactions are sensitive to EDTA.

• Working concentrations (general):

  • Prepare 50–500 ng/µL stocks for routine cloning; 0.5–2 µg/µL for transfection/packaging, depending on cell type and method.

• Electrophoresis buffers (general):

  • TAE (lower ionic strength, better for long fragments, slower run) or TBE (higher buffering capacity) for gel assessment of plasmid integrity.

• Sterility and nuclease control:

  • Use sterile, DNase/RNase-free tubes and tips; if sterile filtration is required, use 0.22 µm low-protein-binding filters. Avoid repeated freeze–thaw.

Note: Any supplied buffer composition for this item is not specified; refer to CoA/Spec Sheet. Recipes above are general laboratory guidance.

Green Alternatives

Green chemistry considerations center on nucleic acid workflows rather than solvent replacement.

• Safer stains (literature/general): Replace ethidium bromide with less hazardous DNA stains (e.g., SYBR Safe, GelRed/GelGreen). Dispose according to local regulations.
• Minimize hazardous waste: Prefer aqueous buffers over phenol–chloroform extractions; use column- or magnetic bead–based purifications to reduce halogenated solvent use.
• Energy efficiency: Consolidate freeze–thaw cycles; use high-efficiency −80 °C freezers and insulated sample boxes to reduce compressor load.
• Plastic reduction: Employ reusable chilled blocks in place of excess dry ice on the bench; choose low-binding, recyclable plastics when compatible with nuclease control.
• Viral packaging alternatives: When feasible, use transient plasmid transfection (non-viral delivery) for short-term assays to avoid BSL‑2 viral waste; weigh against experimental requirements.

Comparison (general):

• Traditional gel extraction: Often uses guanidinium salts and organic solvents. Greener alternative: silica columns with aqueous buffers or enzyme-based PCR cleanup, reducing solvent hazards.
• Ethidium bromide decontamination: Requires special waste handling. Greener alternative: non-mutagenic stains compatible with standard waste streams (verify locally).

Note: The composition of this specific item and included buffers/additives are not specified; refer to CoA/Spec Sheet for any reagent-specific environmental, health, and safety notes.

Pharmaceutical Uses

This item is a research-use-only plasmid DNA reagent and is not an excipient, API, or clinical-grade vector.

• Regulatory status: For research use only (per Product Data). Not intended for diagnostic, therapeutic, or in vivo clinical applications.
• GMP/Pharmacopeia: No GMP, USP/EP, or clinical-grade status is specified for this item; refer to CoA/Spec Sheet if regulatory documentation is needed for non-clinical studies.
• Formulation context (general): In preclinical research settings, plasmids may be formulated with transfection reagents for in vitro use or packaged into viral particles for cell-based assays. Any such use must comply with institutional biosafety rules and remains strictly non-clinical.

Recommendation: If translational or GLP-adjacent work is contemplated, obtain detailed QC documentation (sequence verification, endotoxin levels, sterility/bioburden) and assess vendor change-control and lot traceability. None of these attributes are specified for this item and must be verified independently.

Physical Properties

As a nucleic acid reagent, physical properties depend on formulation and concentration.

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet. (Plasmid MW depends on base-pair length; literature: ~660 Da per bp.)
• Solubility (literature/general): Highly water-soluble in nuclease-free aqueous buffers (e.g., 10 mM Tris-HCl, 0.1–1 mM EDTA, pH ~7.5–8.0) or nuclease-free water. Insoluble in nonpolar organic solvents.
• pKa/logP (not applicable): Classical small-molecule pKa/logP do not apply to polyanionic DNA macromolecules.
• UV properties (literature/general): ss/ds DNA exhibits strong absorbance at 260 nm; typical A260 of 1.0 corresponds to ~50 µg/mL dsDNA; A260/A280 ~1.8 indicates protein-free DNA.
• Density/refractive index/viscosity: Not specified for this item; refer to CoA/Spec Sheet. For dilute aqueous DNA solutions, values approximate those of water (literature/general).
• Melting/boiling point: Not applicable to aqueous DNA solutions; thermal denaturation depends on sequence and ionic strength (literature/general).

Handling note (general): Maintain solutions on ice during bench work to reduce nuclease activity; avoid repeated freeze–thaw to preserve integrity.

Quality and Grades

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

Interpretation for plasmid DNA reagents (general):

• Sequence identity: For sgRNA vectors, quality is commonly established by Sanger or NGS confirmation of the guide insert and junctions. Exact verification methods for this item are not specified.
• Topology and integrity: Supercoiled fraction and absence of nicks are important for high-efficiency transfection and packaging. Agarose gel or capillary electrophoresis profiles are typical QC tools (literature/general).
• Endotoxin content: For cell culture applications, low-endotoxin preparations improve transfection/viability. Specific endotoxin limits are not specified for this item; refer to CoA/Spec Sheet.
• Residual contaminants: Protein, RNA, salts, and solvent carryover can impair downstream performance; A260/280 and A260/230 ratios provide quick assessment (literature/general).
• Antibiotic marker and backbone: The identity of bacterial selection marker and exact backbone features are not specified for this item; consult the CoA/Spec Sheet/plasmid map.

Stabilizers/additives: Not specified for this item; refer to CoA/Spec Sheet.

Recommendation: Request the sequence file (.gb/.fa), QC chromatograms, and endotoxin report if your workflow involves sensitive cells or in vivo models (research use only).

Reaction and Applications

As a lentiviral CRISPR sgRNA plasmid targeting ARG2, this reagent supports genome engineering and functional genomics workflows.

Key applications (literature/general):

• CRISPR knockout/knockdown of ARG2: Co-delivery with Cas9 (or use in Cas9-expressing cells) to generate frameshift-inducing indels at ARG2 loci via NHEJ.
• Lentiviral delivery: Packaging into VSV-G pseudotyped lentiviral particles enables stable delivery to dividing and non-dividing mammalian cells. Requires BSL‑2 practices and appropriate packaging systems.
• Pooled/arrayed screening: Inclusion in libraries or as a single construct for target validation, metabolic pathway interrogation, and synthetic lethality screens.
• Reporter or rescue studies: Pair with ARG2 cDNA constructs (recoded/sgRNA-resistant) for rescue experiments to confirm on-target phenotypes.

Practical considerations (general):

• Guide design: On-target activity and specificity depend on the exact spacer sequence and PAM compatibility (e.g., NGG for SpCas9). Guide sequence for this item is not specified; verify via CoA/sequence file.
• Verification: After editing, confirm ARG2 disruption by T7E1, ICE/TIDE, amplicon NGS, or Western blot/qPCR for downstream effects.
• Controls: Use non-targeting sgRNA and a positive control sgRNA to benchmark editing efficiency.
• DNA quality: High supercoiled fraction and low endotoxin improve transfection/packaging yields.

Tip: For lentiviral use, titrate MOI and include polybrene (as permitted) and appropriate antibiotic/fluorescent selection encoded by the transfer vector (specific marker not specified for this item).

Reaction Conditions

Small-molecule reaction conditions are not applicable. The following are general conditions for common workflows involving sgRNA plasmids (literature/general):

• Bacterial propagation:

  • Transform into chemically competent E. coli (e.g., 30–60 s heat shock at 42 °C) or electrocompetent cells (1.8 kV, 5 ms). Recover in SOC at 37 °C for 45–60 min; plate on appropriate antibiotic (marker not specified for this item).
  • Grow starter culture (3–5 mL), then scale to midiprep/maxiprep; avoid overgrowth to limit nuclease stress.

• Transfection (plasmid delivery):

  • Seed cells to 60–80% confluency. Use lipid/polymer transfection per supplier’s instructions; typical DNA amounts 0.5–2 µg per well (6‑well format). Optimize DNA:reagent ratios; change medium after 6–24 h.

• Lentiviral packaging (BSL‑2):

  • Co-transfect transfer vector with packaging (gag/pol, rev) and envelope (e.g., VSV‑G) plasmids into HEK293T cells. Collect supernatant at 48–72 h; filter (0.45 µm). Titrate virus (p24, RT assay, or functional titer). Transduce target cells at desired MOI with polybrene if permitted; select after 48–72 h.

• Editing assessment:

  • Harvest genomic DNA 48–96 h post-delivery; PCR amplify target; analyze by T7E1, ICE/TIDE, or amplicon NGS. Confirm protein-level effects by immunoblot.

Note: Exact guide sequence, promoter, and selection marker for this item are not specified; refer to CoA/Spec Sheet for construct-specific parameters.

Safety and Handling

• GHS classification and hazard statements: Not specified for this item; refer to SDS. DNA plasmid preparations are generally not classified as hazardous chemicals, but institutional biosafety rules apply.
• Signal word/pictograms: Not specified for this item; refer to SDS.

Biosafety considerations (general, literature):

• Risk group/material type: Non-replicating plasmid DNA. If used to produce lentiviral particles, packaging and transduction work typically requires BSL‑2 containment with appropriate approvals.
• Nuclease contamination: Use clean, nuclease-free techniques and PPE (gloves, lab coat, eye protection). Change gloves frequently; avoid touching common surfaces with contaminated gloves.

Storage incompatibilities and precautions:

• Avoid nucleases, microbial contamination, and repeated freeze–thaw cycles. Use low-retention, DNase/RNase-free plastics. Do not mix with strong oxidizers or DNase-containing reagents.

PPE and engineering controls (general):

• Wear safety glasses, lab coat, and disposable nitrile gloves. Use a certified biosafety cabinet for lentiviral packaging or any aerosol-generating step. Disinfect surfaces with DNA-active agents (e.g., 10% bleach) followed by 70% ethanol.

First-aid overview (general):

• Skin/eye contact: Rinse with water. Seek medical advice if irritation persists. Ingestion/inhalation: Rinse mouth, move to fresh air; seek medical attention as needed. Always defer to site SDS and institutional biosafety procedures.

Solvent Selection

This product is a DNA plasmid; organic solvent selection is not typically applicable. Focus is on aqueous, nuclease-free buffers for dissolution, storage, and handling.

• Compatible solvents (general, literature):
  • Nuclease-free water (dH2O) for short-term use and routine cloning.
  • TE buffer (10 mM Tris-HCl, 0.1–1 mM EDTA, pH 7.5–8.0) for improved long-term stability by chelating divalent cations required by nucleases.
  • Low-salt Tris buffer (e.g., 10 mM Tris, pH 8.0) if downstream enzymology is sensitive to EDTA.
• Incompatible media: Strong acids/bases, chaotropes, and organic solvents (e.g., phenol, chloroform, alcohols) outside of extraction workflows can denature or precipitate DNA.
• Filtration: Use 0.22 µm sterile, low-protein-binding filters for sterilization if required; avoid shear by minimizing pressure.

Selection guidance (general):

• Choose nuclease-free water for immediate cloning, PCR, or Gibson assembly.
• Choose TE for archival stocks, especially for −20/−80 °C storage.
• For transfection into sensitive cells, ensure endotoxin-minimized buffer and isotonicity after dilution into culture medium.

Note: Exact formulation supplied (if any) is not specified for this item; refer to CoA/Spec Sheet.

Storage and Reconstitution

• Storage conditions (item-specific): Store at −80 °C (per Product Data). Avoid repeated freeze–thaw cycles. Shipments are on dry ice packs + cold packs.
• Formulation/appearance: Not specified for this item; refer to CoA/Spec Sheet.

Reconstitution (general, literature):

• If provided lyophilized: Spin tube to collect contents. Add nuclease-free water or TE buffer (10 mM Tris-HCl, pH 7.5–8.0; 0.1–1 mM EDTA) to achieve 50–500 ng/µL (or as required). Mix gently; avoid vigorous vortexing to minimize shearing.
• If provided in solution: Keep on ice during handling. Upon first thaw, aliquot into single-use volumes in DNase/RNase-free tubes.

Storage best practices (general):

• Long-term: Maintain master aliquots at −80 °C as specified. Protect from repeated freeze–thaw; use low-binding tubes.
• Working stocks: Many labs keep short-term working aliquots at −20 °C for convenience; however, defer to −80 °C for long-term stability per Product Data.
• Nuclease control: Use sterile, nuclease-free consumables. Consider TE buffer for archival stability; use water for immediate enzymatic applications.

Labeling and tracking:

• Record lot/aliquot IDs, concentration (A260-based), buffer, and date of first thaw. Verify concentration periodically if used for quantitative transfections.

Research use only: As stated in Product Data; not for diagnostic or therapeutic use.

Structure and Identity

This product is a DNA plasmid reagent: a lentiviral CRISPR single‑guide RNA construct designed to target the ARG2 gene (Arginase 2), supplied under the name pLenti-ARG2-sgRNA.

• Item-specific identifiers

  • SKU: P743869
  • Product name: pLenti-ARG2-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 to plasmid DNA constructs; not assigned.

• Structural features (general, literature)

  • Macromolecule type: double-stranded circular DNA plasmid suitable for lentiviral packaging (literature/general for lentiviral sgRNA vectors).
  • Typical elements: U6 or H1 RNA polymerase III promoter driving the sgRNA; sgRNA scaffold sequence compatible with a Cas nuclease (commonly SpCas9; literature/general). Lentiviral backbone elements (e.g., 5'/3' LTRs, Ψ packaging signal) are typical of pLenti formats (literature/general).
  • Target: sgRNA is designed to direct Cas nuclease to ARG2 genomic loci through base pairing to the protospacer adjacent to an appropriate PAM (e.g., NGG for SpCas9; literature/general). Specific guide sequence is not specified for this item; refer to CoA/Spec Sheet.

• 2D description in words (general)

  • Plasmid maps depict circular DNA with labeled functional cassettes: promoter → sgRNA spacer → tracr/scaffold; antibiotic resistance marker for bacterial propagation; origin of replication; lentiviral elements for packaging (literature/general).

Note: Exact plasmid length (bp), antibiotic selection, promoter choice, and guide sequence are not specified for this item; refer to CoA/Spec Sheet.

Synthetic Utility

Classical small-molecule synthetic utility does not apply. However, from a molecular biology and genetic engineering perspective, pLenti-ARG2-sgRNA serves as a modular genetic construct.

• Functional modules (general, literature):

  • sgRNA expression cassette under a Pol III promoter (e.g., U6/H1) directing Cas nuclease to ARG2.
  • Lentiviral transfer backbone with packaging signals enabling incorporation into pseudotyped lentiviral particles for stable delivery.
  • Bacterial origin of replication and antibiotic resistance for propagation in E. coli.

• Typical manipulations:

  • Subcloning the sgRNA spacer: Oligo annealing and ligation or Golden Gate/Gibson Assembly to swap spacers; verify by Sanger sequencing.
  • Cassette exchange: Replace promoters or add selection markers via standard restriction/assembly methods.
  • Dual-guide or multiplexing: Insert additional guide cassettes for combinatorial targeting (literature/general).

• Retrosynthetic value (general):

  • The plasmid can act as a donor for sgRNA cassettes into alternative vectors (e.g., PiggyBac, AAV) when backbone constraints require different delivery modalities.

Note: Exact restriction sites, MCS layout, antibiotic marker, and vector length are not specified for this item; refer to CoA/Spec Sheet/plasmid map.

Target Specificity

• Intended target: ARG2 (Arginase 2) genomic loci.
• sgRNA spacer sequence and PAM compatibility: Not specified for this item; refer to CoA/Spec Sheet/sequence file.

General considerations for CRISPR specificity (literature/general):

• PAM dependence: For SpCas9 systems, on-target cleavage requires an NGG PAM immediately downstream of the protospacer on the non-target strand.
• Mismatch tolerance: Seed region mismatches (proximal to PAM) strongly reduce binding, whereas distal mismatches may be tolerated—evaluate computationally and empirically.
• Off-target risk mitigation: Use high-fidelity Cas9 variants, truncated guides (17–18 nt), chemically modified sgRNAs, or paired nickases where appropriate.
• In silico design: Employ genome-wide scoring tools (e.g., CFD scores) and filter against exonic off-targets in genes critical to the model system.
• Empirical confirmation: Validate with amplicon NGS and rescue experiments (re-expression of sgRNA-resistant ARG2) to attribute phenotypes to on-target edits.

Note: Clone ID, backbone identity, and any selectable markers are not provided here. Target performance ultimately depends on the exact spacer and cell context; consult the CoA/Spec Sheet for construct-specific details.