pLenti-ACTB-sgRNA

Cat. No.: P743598

Spécifications

Product Name

pLenti-ACTB-sgRNA

Product Line

Produits biologiques

Stockage et expédition

Conditions de stockage de stockage

Store at -80°C

Expédié en

Dry ice packs + Cold packs

Stabilité et stockage

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

Application Protocols

No manufacturer‑validated protocols, tested applications, or recommended dilutions are provided for this item.

General workflow outlines (literature/guidance; adapt to your system):

• Plasmid verification: Sequence across U6 promoter → spacer insert → scaffold; confirm backbone integrity.
• Lentiviral production (BSL‑2): Co‑transfect with packaging plasmids; collect virus at 48–72 h; filter (0.45 µm); optionally concentrate by PEG precipitation or ultracentrifugation.
• Transduction and selection: Transduce target cells; select per backbone marker (e.g., puromycin) if present; expand monoclonal populations for genotyping.
• Editing assessment: Perform PCR of ACTB locus; analyze by ICE/TIDE or NGS; validate at protein level by immunoblotting for β‑actin or by phalloidin staining for F‑actin morphology changes.

Because item‑specific parameters (vector map, selection marker, promoter) are not specified, users should refer to the CoA/sequence file and adapt standard CRISPR‑lenti protocols accordingly.

Biological Roles

Target gene context (general biology):

• ACTB encodes β‑actin, a highly conserved, abundant cytoskeletal protein critical for cell structure, motility, intracellular trafficking, and cytokinesis. Actin monomers (G‑actin) polymerize into filaments (F‑actin), dynamically remodeling in response to signaling pathways (Rho family GTPases, PI3K) and ATP hydrolysis.
• β‑actin participates in formation of the cortical cytoskeleton, stress fibers, and adhesion complexes. It also influences gene expression via mechanotransduction and chromatin interactions.
• Because of its ubiquitous expression, ACTB is often used as a housekeeping reference in molecular assays; however, CRISPR disruption can be deleterious and requires careful titration and rescue strategies in essentiality contexts (design consideration).

Use of an ACTB‑targeting sgRNA (general, literature):

• Enables functional studies of cytoskeletal dynamics, validation of CRISPR editing workflows, and benchmarking of delivery/editing efficiency.
• In CRISPRi/CRISPRa applications, modulation of ACTB transcript levels can probe dosage effects on cellular mechanics.

Notes specific to this item:

• This product provides the sgRNA expression element in a lentiviral plasmid backbone. Exact spacer sequence, PAM, promoter, and selectable marker are not specified; consult the CoA/sequence map for definitive design details before experimental planning.

Buffer Applications

Not typically applicable. This product is a plasmid DNA reagent, not a buffering agent. For routine handling, nuclease‑free water or Tris‑EDTA buffers are used to stabilize DNA (general, literature). Refer to Storage & Reconstitution for practical guidance on diluents and handling.

Green Alternatives

Green chemistry substitution is not directly applicable to a plasmid DNA reagent. Environmental impact is driven more by workflow choices (plasticware, solvents in DNA purification, and energy for cold storage) than by the reagent itself.

Sustainable practice suggestions (general, literature):

• Minimize single‑use plastics by consolidating prep volumes and using recyclable tip boxes where permitted.
• Prefer ethanol/isospropanol precipitation or spin‑column methods that reduce hazardous organic solvent use compared to phenol–chloroform extractions.
• Optimize freezer management: store working aliquots at −20 °C (short term) and archive at −80 °C to reduce door‑open time; use frost‑free practices and inventory systems to limit energy waste.
• Shipments: Coordinate consolidated orders to reduce packaging waste and cold‑chain shipments.

These recommendations are general and do not alter the item’s specifications or use conditions.

Pharmaceutical Uses

Not applicable. This lentiviral plasmid is supplied strictly for research use only (non‑GMP). It is not intended for diagnostic, therapeutic, or human/animal administration. No pharmacopeial status, excipient role, or manufacturing use is claimed or specified for this item.

Physical Properties

As a plasmid DNA reagent, conventional small‑molecule physical constants do not apply. Item‑specific solution properties are not provided.

• Physical state: Not specified for this item; refer to CoA/Spec Sheet (commonly supplied in aqueous buffer or lyophilized DNA).
• Concentration (A260‑based) and purity (A260/280, A260/230): Not specified for this item; refer to CoA/Spec Sheet.
• Sequence length (bp) and backbone map: Not specified for this item; refer to CoA/Spec Sheet.
• pH and buffer composition: Not specified for this item; refer to CoA/Spec Sheet.

General/literature properties for plasmid DNA solutions (for context only):

• DNA absorbs at 260 nm (1 A260 ≈ 50 µg/mL for dsDNA; literature).
• Typical storage buffers contain 10 mM Tris‑HCl, 0.1–1 mM EDTA, pH 7.5–8.5 (literature).
• Plasmid DNA is water‑soluble; solubility influenced by ionic strength and counterions (literature).

Handling implications:

• Avoid repeated freeze–thaw cycles to maintain supercoiled fraction and cloning/packaging efficiency (general guidance).
• Gentle mixing is preferred (no vortexing) to minimize shearing of high‑MW DNA. Use low‑adhesion tubes and pipette tips.

Any specific volumetric, density, refractive index, logP, melting/boiling points: Not applicable to plasmid DNA and not specified for this item.

Quality and Grades

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

Interpretation and expectations for plasmid DNA reagents (general guidance):

• Quality attributes commonly reported include supercoiled percentage, A260/280 and A260/230 ratios, endotoxin level (for cell culture/transfection), sterility (no bacterial/fungal growth), and sequence verification of the sgRNA spacer and backbone junctions.
• Typical QC methods: restriction digest profiling, capillary electrophoresis or agarose gel to assess topology, Sanger sequencing across the U6 promoter‑sgRNA insert, and optionally full plasmid NGS for critical applications.
• Endotoxin: For packaging or transfection into sensitive primary cells, low‑endotoxin prep is preferred. If not specified, consider performing an endotoxin clean‑up prior to use.
• Stabilizers: Not specified for this item; refer to CoA/Spec Sheet. DNA preparations may include Tris/EDTA; EDTA chelates Mg2+ to inhibit nucleases.

How to use grade information:

• For high‑efficiency lentiviral production, choose lots with high supercoiled content and low endotoxin.
• For cloning or PCR template use, standard miniprep‑level purity may suffice, but verify insert integrity by sequencing.

Documentation:

• Request the sequence map/GenBank file, CoA, and SDS for lot‑resolved quality details.

Reaction and Applications

This pLenti‑ACTB‑sgRNA plasmid is intended for CRISPR guide RNA expression targeting the ACTB (β‑actin) locus. Typical laboratory applications (general, literature) include:

• Lentiviral packaging and cell transduction:
  • Co‑transfect HEK293T or similar producer cells with this sgRNA plasmid plus compatible packaging and envelope plasmids to generate VSV‑G‑pseudotyped lentiviral particles.
  • Transduce target cells to deliver the sgRNA cassette stably; selection markers and MOI optimization depend on the backbone (not specified for this item).
• CRISPR experiments:
  • Pair the sgRNA with a Cas nuclease source (e.g., SpCas9, SaCas9, or catalytically dead Cas9 fusions) provided via a separate plasmid, integrated transgene, or RNP. This item provides only the sgRNA component; Cas source is not specified.
  • Use for gene knockout (NHEJ indels), knock‑in (HDR with donor template), CRISPRi/a (with dCas9‑KRAB/VPR) targeting ACTB.
• Functional genomics:
  • Validate cytoskeletal dependencies, cell morphology pathways, or assay transduction/transfection efficiencies using ACTB as a control target (experimental design varies; literature guidance).

Practical tips:

• Verify the exact spacer sequence and PAM compatibility with your Cas variant; confirm absence of common SNPs in your cell line.
• Use multiple independent guides to mitigate guide‑specific bias; include non‑targeting and safe‑harbor controls.
• Quantify editing via TIDE/ICE, amplicon NGS, or Western blot/qPCR for downstream effects (assays not provided with this item).

Reaction Conditions

Chemical reaction conditions are not applicable. For molecular biology workflows involving this plasmid (general, literature):

• Transfection for lentiviral production: Seed HEK293T cells to 60–80% confluence; transfect with the sgRNA plasmid plus packaging and envelope plasmids using a lipid reagent or PEI. Typical DNA:reagent ratios follow vendor guidelines (e.g., 1:3 w/w for lipids), with Opti‑MEM or serum‑free medium during complexation. Replace medium 6–12 h post‑transfection; harvest viral supernatant at 48–72 h.
• Cell transduction: Add filtered viral supernatant with polybrene (e.g., 4–8 µg/mL; cell‑line dependent) and spinoculate if needed. Select transduced cells according to the backbone’s selectable marker (not specified for this item).
• Editing readout: Extract genomic DNA 48–96 h after Cas9 expression; amplify target locus and assess indels via T7E1, ICE/TIDE, or amplicon NGS. Protein‑level effects may require 3–7 days depending on protein turnover.

Note: Exact conditions vary with cell type, backbone features, and transfection system. Refer to your institution’s BSL‑2 policies for lentiviral work. This section is general guidance and not item‑specific specifications.

Safety and Handling

GHS/SDS details for this specific item are not provided in the product data. Always consult the product SDS and your institutional biosafety program before use.

• GHS signal word, H‑statements, pictograms: Not specified for this item; refer to SDS.
• Biosafety considerations (general, literature):
  • Plasmid DNA itself is non‑replicative outside host cells. However, pLenti backbones are designed for lentiviral packaging. Lentiviral particle production requires BSL‑2 practices (or higher per institutional policy), use of third/fourth‑generation split packaging systems, and appropriate engineering controls (biological safety cabinet, sealed rotors).
  • Do not handle packaging or transduction steps in open bench environments; follow local IBC approvals.
• PPE: Laboratory coat, disposable nitrile gloves, and eye protection are recommended. Use aerosol‑resistant tips.
• Incompatibilities: Avoid nucleases (DNase) and nuclease‑contaminated consumables; use sterile, nuclease‑free water/buffers. Avoid strong oxidants/UV exposure that can nick DNA.
• First aid (general): In case of accidental exposure to lentiviral particles (if generated), follow institutional exposure protocols. For skin/eye contact with solutions, rinse with water and seek medical evaluation per SDS.
• Decontamination: Surfaces and waste potentially containing lentiviral particles should be treated with freshly prepared 0.5–1% sodium hypochlorite (10–20 min contact) or appropriate disinfectant, followed by autoclaving where applicable (literature guidance).

Solvent Selection

This product is a plasmid DNA reagent and is typically supplied in an aqueous buffer. Organic solvent selection is not applicable.

• Solubility context: Double‑stranded plasmid DNA is readily soluble in nuclease‑free water or low‑ionic‑strength Tris/EDTA buffers (general, literature). Avoid alcohols and strong chaotropes unless performing extraction/purification steps.
• Practical guidance:
  • For transfection, dilute in sterile, nuclease‑free water or the buffer recommended by the transfection reagent vendor.
  • For cloning/PCR, water or 10 mM Tris‑HCl, pH ~8.0 is typical to maintain stability.

If an organic solvent‑based workflow is needed (e.g., phenol–chloroform extraction), follow molecular biology protocols; such steps are outside routine handling of a ready‑to‑use plasmid DNA reagent.

Storage and Reconstitution

• Storage conditions (item‑specific): Store at −80 °C. Shipped on dry ice packs + cold packs to maintain cold chain.
• Thawing: Thaw on ice. Mix gently by flicking or slow pipetting; avoid vortexing to preserve supercoiled DNA.
• Aliquoting: Prepare single‑use aliquots in sterile, nuclease‑free, low‑adhesion tubes to minimize freeze–thaw cycles. Keep working aliquots on ice during setup.
• Buffer/diluent: Not specified for this item; refer to CoA/Spec Sheet. If buffer is not indicated, nuclease‑free water or 10 mM Tris‑HCl, pH 7.5–8.5 with 0.1–1 mM EDTA are commonly used for plasmid DNA (general, literature). Avoid chelators in reactions requiring divalent cations unless subsequently diluted.
• Short‑term storage: For frequent use, −20 °C storage of aliquots is common; avoid repeated freeze–thaw. For long‑term archiving, maintain a master stock at −80 °C (item‑specific requirement).
• Sterility: Work in a clean area with nuclease‑free consumables. If contamination is suspected, re‑purify by spin column and re‑quantify by A260.
• Documentation: Record lot number, concentration, and number of freeze–thaw events. Retain the sequence map/CoA for reference.

Research use only: Not for human or animal use. Follow institutional biosafety procedures, especially if generating lentiviral particles.

Structure and Identity

This product is a recombinant DNA reagent: a lentiviral plasmid carrying a single‑guide RNA (sgRNA) targeting the ACTB (β‑actin) gene.

• SKU: P743598
• Product Name: pLenti-ACTB-sgRNA
• Type: Lentiviral plasmid (non‑coding RNA expression cassette for CRISPR)
• Molecular formula: Not applicable to plasmids (polymeric DNA); item‑specific composition not specified.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.
• SMILES/InChI/InChIKey: Not applicable to large nucleic acids; not provided.
• CAS/CID: Not applicable/not assigned for plasmid constructs.

2D/architectural description (general, literature):

• Typical pLenti sgRNA vectors comprise: 5′ and 3′ LTRs, packaging signal (ψ), Rev‑response element (RRE), central polypurine tract (cPPT), and a U6 (or H1) promoter driving the sgRNA scaffold with a 20‑nt ACTB‑targeting spacer. Selection markers (e.g., antibiotic resistance) and reporter cassettes may be present depending on backbone. Specific backbone features for this item are not specified; consult the sequence map/annotated GenBank from the CoA.

Functional groupings (general):

• Guide region: 20 nt spacer complementary to ACTB target sequence (PAM‑proximal site selection depends on the paired Cas variant; not specified here).
• Scaffold: tracrRNA hairpins for Cas loading and target recognition.
• Lentiviral elements: enable packaging into VSV‑G pseudotyped particles when co‑transfected with packaging plasmids (performed under appropriate biosafety conditions).

Synthetic Utility

Classical small‑molecule synthetic utility does not apply to plasmid DNA. From a molecular biology perspective (general):

• Cloning/editing utility: The sgRNA cassette can be re‑cloned into alternative backbones, swapped for different promoters, or multiplexed using Golden Gate or Gibson Assembly strategies.
• Modular design: Lentiviral elements (LTRs, ψ, RRE, cPPT, WPRE) facilitate packaging and stable integration when combined with packaging systems; regulatory elements can be recombined to tailor expression context.
• Retrosynthetic planning (molecular biology): Backbone linearization via restriction sites or PCR, followed by insertion of new spacer oligos, enables rapid retargeting to new genes while retaining lentiviral delivery capability.

Item‑specific restriction maps, sequence features, and cloning sites are not specified; consult the CoA/sequence map for exact synthetic workflows.

Target Specificity

Item‑specific targeting details are not provided in the product data.

• Target gene: ACTB (β‑actin) — inferred from the product name; exact spacer sequence, strand orientation, PAM compatibility, and species ortholog are not specified for this item; refer to CoA/Spec Sheet.
• Antigen/epitope/clone/isotype: Not applicable (this is not an antibody despite the category path label).
• Cross‑reactivity/off‑targets: Not specified for this item; users should assess potential off‑target sites bioinformatically based on the actual spacer sequence once provided.