pLenti-CDK5RAP2-sgRNA

Cat. No.: P744759

Specifications

Product Name

pLenti-CDK5RAP2-sgRNA

Product Line

biological_products

Storage and Shipping

Storage

Store at -80°C

Shipped In

Dry ice packs + Cold packs

Stability And Storage

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

Application Protocols

Note: The following are generalized protocols for pLenti sgRNA workflows; adapt to your backbone, cell type, and institutional SOPs. Item-specific sequence, promoter, marker, and buffer are not specified for this product.

• A. Cloning/verification of CDK5RAP2 sgRNA (if cloning required)

  1. Design two 20‑nt oligos with appropriate overhangs for your cloning method (e.g., BsmBI for Golden Gate). 2) Anneal/ligate into the backbone. 3) Transform into high‑efficiency E. coli; screen colonies by PCR. 4) Verify insert and scaffold by Sanger sequencing.

• B. Preparation for lentiviral packaging

  1. Grow HEK293T cells to ~70% confluence. 2) Prepare DNA: packaging + envelope + pLenti‑CDK5RAP2‑sgRNA at recommended ratios per transfection reagent SOP. 3) Complex DNA with transfection reagent in serum‑free medium, incubate, then add to cells. 4) Replace medium after 6–24 h.

• C. Viral harvest and transduction

  1. Collect supernatant at 48 and 72 h; clarify and, if needed, concentrate. 2) Transduce target cells with appropriate MOI in presence/absence of polybrene as cell‑line compatible. 3) Change medium after 12–24 h.

• D. Selection and validation

  1. Apply antibiotic or reporter-based selection if supported by the backbone (not specified for this item). 2) Assess editing at 72–120 h by TIDE/ICE or amplicon NGS. 3) Confirm protein loss by immunoblot/IF; perform rescue where necessary.

• E. Clonal isolation (optional)

  • Single‑cell clone by limiting dilution or FACS; expand and genotype to identify biallelic knockouts.

Biological Roles

Focus: Target gene CDK5RAP2 (CDK5 regulatory subunit-associated protein 2), the intended genomic target of the sgRNA in this construct.

• Literature/general biology (not item-specific)

  • Cellular function: CDK5RAP2 is a conserved centrosomal/pericentriolar material (PCM) protein implicated in microtubule nucleation and anchoring, γ‑tubulin ring complex (γ‑TuRC) recruitment, centrosome cohesion, and spindle assembly checkpoint signaling.
  • Developmental relevance: Loss-of-function mutations in CDK5RAP2 are associated with primary microcephaly (MCPH3) in humans, highlighting its role in neural progenitor proliferation and mitotic fidelity (literature).
  • Cell‑cycle dynamics: Levels and localization of CDK5RAP2 are regulated through the cell cycle, coordinating centrosome maturation at G2/M and ensuring accurate chromosome segregation.
  • Interactions: Interfaces with PCM proteins (e.g., pericentrin), dynein/dynactin transport machinery, and γ‑TuRC components for microtubule organization (literature).
  • Experimental perturbation: CRISPR-mediated disruption produces phenotypes such as abnormal centrosome number, disorganized spindles, mitotic delay, and altered cell polarity, which can be quantified by high‑content imaging and live‑cell microscopy.

• Scope note

  • The above summarizes general, peer‑reviewed biology of CDK5RAP2. This product’s exact spacer sequence and on‑target cut site within CDK5RAP2 are not specified for this item; refer to CoA/Spec Sheet for construct‑specific details, if provided.

Buffer Applications

Applicability note: As a nucleic acid reagent, buffer choice impacts stability and downstream performance rather than pH‑dependent chemical equilibria as in small‑molecule buffers.

• Product Data (item-specific)

  • Supplied buffer/composition: Not specified for this item; refer to CoA/Spec Sheet.

• Literature/general guidance

  • Long-term storage: TE (10 mM Tris‑HCl, 0.1–1 mM EDTA, pH 7.5–8.0) is widely used to protect DNA from nuclease degradation; store at −20 to −80°C in aliquots to minimize freeze–thaw.
  • Enzymatic reactions: Use low‑salt, EDTA‑free buffers or nuclease‑free water for restriction digestion, ligation, Gibson/Golden Gate assembly, and PCR to avoid divalent cation chelation that can inhibit enzymes.
  • Transfection/electroporation: For chemical transfection, follow reagent‑specific complexation buffers (often serum‑free media). For electroporation, exchange into low‑conductivity, proprietary electroporation buffers or nuclease‑free water as recommended by the instrument manufacturer.
  • Viral packaging: Prepare DNA mixes in sterile, serum‑free medium compatible with the transfection reagent used in HEK293T or similar producer cells; maintain physiological pH (~7.2–7.4) and osmolarity.

• Practical tips

  • Filter-sterilize (0.22 µm) working buffers; use nuclease‑free plastics; include RNase/DNase‑free certification.
  • Avoid repeated freeze–thaw; use small, single‑use aliquots.

Green Alternatives

Although this is a genetic reagent rather than a solvent or bulk chemical, greener choices can be made in workflows using pLenti sgRNA constructs.

• General opportunities (literature/guidance)

  • Solvent minimization: Favor aqueous buffers over chlorinated/oxygenated organics during cloning and purification. Use ethanol/IPA judiciously and capture for recycling when possible.
  • Enzymatic assembly: Gibson, Golden Gate, or HiFi assemblies proceed in aqueous media at mild temperatures, reducing energy input and hazardous waste versus older multi‑step cloning with large solvent use.
  • Endotoxin‑free prep kits: Choose kits that avoid phenol/chloroform extractions and employ membrane or magnetic‑bead technologies to reduce hazardous waste streams.
  • Energy efficiency: Consolidate incubations and use room‑temperature stable enzymes where validated to lower refrigeration/freezer door‑open time and energy consumption.

• Tradeoffs

  • EDTA-containing buffers improve DNA stability but may necessitate desalting before electroporation, adding a processing step.
  • Phenol‑free plasmid preps are safer but occasionally yield lower DNA in challenging strains; optimization may be required.

• Simple comparison (general)

  • Phenol/chloroform extraction: high purity but hazardous waste; avoid when possible.
  • Column/bead purification: safer, aqueous, scalable; lower environmental burden; comparable purity for most applications.

Pharmaceutical Uses

This product is designated For research use only and is not intended for human or veterinary use, diagnostics, or therapeutic applications.

• Applicability

  • As a research-grade lentiviral sgRNA plasmid, it is not an excipient, API, or GMP material. No pharmacopeial monograph applies.

• Literature/general context (not item-specific; for background only)

  • Similar vector formats under GMP can be components of manufacturing processes for cell/gene therapy research; however, transitioning from research to clinical grade requires extensive re‑engineering of quality systems, documentation, and release testing (e.g., identity, purity, sterility, endotoxin, residuals, adventitious agents). These considerations do not apply to this research‑use item.

• Guidance

  • Do not use in humans. For any translational intent, source GMP‑compliant materials and follow regulatory guidance (ICH, USP <1043>) and institutional oversight.

• Item-specific status

  • No clinical or formulation uses are specified for this item; refer to CoA/Spec Sheet for any additional compliance statements.

Physical Properties

• Product Data (item-specific)

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

• Applicability to macromolecular DNA constructs

  • Classical small-molecule metrics (BP, MP, logP, refractive index) are not applicable to plasmid DNA.

• Literature/general properties for plasmid DNA (not item-specific)

  • Physical state: typically supplied as an aqueous solution (nuclease‑free water or low-salt buffer) or as a dried film/lyophilizate.
  • Solubility: plasmid DNA is highly soluble in aqueous buffers; insoluble in nonpolar organic solvents.
  • UV absorbance: A260 used for quantification; A260/A280 ~1.8–2.0 indicates low protein contamination (literature).
  • pH compatibility: stable across pH ~7–8.5 in low‑salt buffers (literature). TE buffer (10 mM Tris‑HCl, 0.1–1 mM EDTA, pH 7.5–8.0) commonly used to protect from nuclease‑mediated degradation.
  • Stability considerations: sensitive to nucleases; repeated freeze–thaw cycles can shear or reduce supercoiling. Supercoiled topology promotes high transfection and packaging performance (literature).

• Practical notes

  • If supplied frozen, thaw on ice and mix gently (avoid vortexing to preserve supercoiling).
  • For concentration determination, use A260 (1 A260 ≈ 50 µg/mL for dsDNA; literature). These are general reference values, not specifications for this item.

Quality and Grades

• Product Data (item-specific)

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

• Interpreting quality for plasmid/viral vector reagents (literature/general)

  • Sequence identity: Verification by Sanger or NGS of the sgRNA spacer, scaffold, and backbone junctions is critical. Lot-specific sequence confirmation ensures target fidelity and avoids silent mutations affecting folding.
  • Topology and integrity: Supercoiled content and low nicked/linear fractions favor high transfection and packaging efficiency. Agarose gel or capillary electrophoresis can assess topology.
  • Endotoxin levels: For mammalian cell transfection or viral packaging, low endotoxin DNA is strongly preferred (≤10 EU/µg DNA often targeted in practice; literature). Endotoxin reduction improves cell health and titer.
  • Residual contaminants: Assess A260/280 and A260/230, and, when applicable, RNase/protein carryover. For viral stocks, determine functional titer (TU/mL) and absence of replication‑competent lentivirus (RCL) per institutional policy (general guidance).
  • Stabilizers/buffers: Presence of EDTA (e.g., TE) can inhibit some enzymatic steps; nuclease‑free water is often chosen for direct transfection. If stabilizers are present, they should be disclosed on the CoA.

• Documentation

  • For this item, any specific acceptance criteria (sequence, purity, endotoxin, buffer composition, titer) are not specified; consult the CoA/Spec Sheet for definitive quality information.

Reaction and Applications

• Product Data (item-specific)

  • Manufacturer Applications: Not specified in the catalog entry beyond the name; refer to CoA/Spec Sheet.

• Literature/general applications of pLenti sgRNA constructs (not item-specific)

  • CRISPR knockout/knockdown: Express an sgRNA targeting CDK5RAP2 to recruit Cas9 (or other Cas nucleases) for DSB induction and error‑prone repair (NHEJ/MMEJ), generating frameshifts and functional knockouts.
  • Stable cell line generation: Lentiviral delivery enables integration of sgRNA cassettes for durable expression across dividing and non‑dividing cells; suitable for pooled or arrayed screens.
  • Functional genomics: Assess roles of CDK5RAP2 in centrosome cohesion, microtubule nucleation, spindle assembly, and cell cycle control by perturbation and phenotypic readouts (imaging, FACS, -omics).
  • Rescue/epistasis studies: Combine with cDNA re‑expression (CRISPR‑resistant constructs) to validate on‑target phenotypes, or pair with additional guides for synthetic lethality/synergy analyses.
  • Tagging/base editing (if compatible): Swap Cas nuclease (e.g., dCas9 effectors, base/prime editors) with the same sgRNA spacer to modulate transcription or introduce precise edits at the CDK5RAP2 locus (general concept; backbone‑dependent).

• Practical tips (general)

  • Guide design: Choose 20‑nt protospacers adjacent to the appropriate PAM for your nuclease (e.g., NGG for SpCas9). Use established tools to minimize off‑targets and maximize on‑target cutting scores.
  • Validation: Confirm indels by TIDE/ICE, amplicon NGS, or targeted cloning/sequencing; assess protein loss by immunoblot or immunofluorescence.

Reaction Conditions

Applicability: Interpreted here as conditions for typical use—transfection, lentiviral packaging, and genome editing—rather than small‑molecule reactions.

• General conditions for lentiviral packaging (literature; not item-specific)

  • Cells: HEK293T or derivative producer lines at 60–80% confluence.
  • Plasmids: sgRNA vector co‑transfected with packaging (gag/pol, rev) and envelope (e.g., VSV‑G) plasmids.
  • Transfection: PEI, calcium phosphate, or lipid-based reagents in serum‑free medium for complexation; replace medium after 6–24 h per reagent SOP.
  • Harvest: Collect viral supernatant at 24–72 h post‑transfection; clarify by centrifugation/filtration; concentrate if needed (PEG/ultracentrifugation) following BSL‑2 procedures.

• General conditions for cell transduction/editing (literature)

  • Target cells: Plate to 30–70% confluence; transduce with appropriate MOI; optional polybrene (hexadimethrine bromide) at 4–8 µg/mL to enhance entry (cell line dependent).
  • Selection: Apply antibiotic/marker selection if the backbone includes selectable markers (not specified for this item) and/or enrich edited cells by FACS if reporters are present.
  • Editing window: Analyze indels 48–96 h after Cas9 expression; expand clones for clonal knockouts as needed.

• Validation and QC

  • Measure editing by TIDE/ICE or amplicon NGS; assess off‑targets for critical applications.

Note: Exact reagent ratios, volumes, selectable markers, and promoters for this item are not specified; follow your institutional SOPs and consult the CoA/Spec Sheet.

Safety and Handling

• Product Data (item-specific)

  • Hazard information (Signal Word, H‑Statements, GHS Classification, Pictograms): Not specified for this item; refer to SDS.
  • Research Use: For research use only.
  • Storage: Store at −80°C (per product data). Shipped on dry ice packs + cold packs.

• General laboratory safety (literature/best practice; not item-specific)

  • Biosafety: Plasmid DNA encoding sgRNA is non-infectious; handle under standard BSL‑1 molecular biology practices. If used to generate lentiviral particles, all downstream work with packaged virus requires BSL‑2/BSL‑2+ containment and institutionally approved SOPs.
  • PPE: laboratory coat, disposable gloves, and safety glasses. Use nuclease‑free technique to avoid contamination/degradation.
  • Incompatibilities: Avoid nucleases (DNase), microbial contamination, and prolonged exposure to elevated temperature. Do not mix with strong oxidants or intercalating dyes outside of appropriate waste streams.
  • First aid (general): In case of skin/eye contact with solutions containing DNA and common buffers, rinse with water. If exposure involves auxiliary reagents (e.g., ethidium bromide, chloroform, PEI, transfection reagents), follow the specific SDS for those chemicals.
  • Waste: Dispose of biological and chemical wastes according to local regulations. Decontaminate surfaces with 10% bleach followed by 70% ethanol when working with lentiviral packaging workflows.

Always consult the SDS and institutional biosafety guidelines for authoritative hazard and handling requirements.

Solvent Selection

Applicability note: As a plasmid DNA/sgRNA construct, traditional solvent selection paradigms for small molecules do not apply. Focus instead on aqueous buffer choice for handling and applications.

• Product Data (item-specific)

  • Formulation/buffer: Not specified for this item; refer to CoA/Spec Sheet.

• Literature/general guidance for DNA constructs

  • Preferred medium: nuclease‑free water or low‑salt buffers (e.g., 10 mM Tris‑HCl, pH 7.5–8.5). EDTA (0.1–1 mM) helps chelate divalent cations and reduce nuclease activity but may inhibit downstream enzymes; choose based on application.
  • Cell delivery: For transient transfection, dilute DNA into serum‑free medium compatible with your transfection reagent (e.g., Opti‑MEM) per reagent instructions. For lentiviral packaging, the DNA mixture is prepared in sterile, serum‑free medium or buffer suitable for the transfection chemistry used in HEK293T cells.
  • Long‑term storage: Aliquot into nuclease‑free water or TE; avoid repeated freeze‑thaw cycles.

• Comparison (general)

  • Water (nuclease‑free): maximizes compatibility with enzymes and transfection; less chemical protection vs TE.
  • TE buffer: enhanced stability against nucleases; may require desalting/exchange before certain enzymatic reactions or electroporation.

Always defer to the item’s CoA/Spec Sheet for the actual supplied buffer and any stabilizers.

Storage and Reconstitution

• Product Data (item-specific)

  • Storage Conditions: Store at −80°C.
  • Shipped In: Dry ice packs + Cold packs.
  • Supplied concentration/formulation: Not specified for this item; refer to CoA/Spec Sheet.

• General guidance (not item-specific)

  • Upon receipt: Keep frozen; minimize time out of cold chain. If solution, thaw on ice; gently mix by slow inversion; avoid vigorous vortexing to preserve supercoiling/topology.
  • Aliquoting: Prepare single‑use aliquots in nuclease‑free tubes to avoid repeated freeze–thaw cycles. Typical working aliquots are sized for one transfection or packaging reaction.
  • Storage buffers: Nuclease‑free water or TE (10 mM Tris‑HCl, 0.1–1 mM EDTA, pH 7.5–8.0) are commonly used for DNA stability. EDTA may inhibit downstream enzymatic steps; exchange buffer if necessary before electroporation or enzyme‑dependent workflows.
  • Reconstitution (if lyophilized): Not specified for this item; refer to CoA/Spec Sheet. General practice is to reconstitute to 50–200 ng/µL in nuclease‑free water or buffer, then quantify by A260.
  • Handling: Work in a clean, DNase‑free environment. Use filter tips and certified nuclease‑free consumables. For lentiviral stocks (if generated downstream), store at −80°C in small aliquots and avoid freeze–thaw; handle under BSL‑2.

• Stability note

  • Long‑term integrity depends on temperature control, buffer composition, and avoidance of nucleases. For definitive stability/titer (if applicable), consult the item’s CoA/Spec Sheet.

Structure and Identity

Brief description: pLenti-CDK5RAP2-sgRNA is a lentiviral CRISPR guide RNA construct designed for gene editing of the human CDK5RAP2 locus via RNA-guided Cas nuclease systems.

• Product Data (item-specific)

  • SKU: P744759
  • Product Name: pLenti-CDK5RAP2-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.
  • SMILES/InChI/InChIKey/CAS/CID: Not applicable for plasmid DNA; not specified for this item; refer to CoA/Spec Sheet.

• Literature/general description of structure (not item-specific; typical of pLenti sgRNA vectors)

  • Macromolecule type: circular double‑stranded DNA plasmid (lentiviral backbone) carrying a single-guide RNA (sgRNA) expression cassette targeting CDK5RAP2.
  • Typical functional modules: 5′-LTR/3′-LTR (or SIN LTRs), packaging signal (Ψ), bacterial origin of replication (e.g., ColE1), antibiotic resistance marker (e.g., AmpR or KanR), and an RNA polymerase III promoter (often U6 or H1) driving the sgRNA, which comprises a 20-nt spacer (CDK5RAP2-targeting) fused to a constant gRNA scaffold.
  • Functional groups/features: nucleic acid polymer with phosphodiester backbone; includes promoter regions, selection markers, and lentiviral elements for packaging into pseudotyped particles in producer cells.
  • 2D “word” structure: circular plasmid map with discrete annotated regions—bacterial propagation region, mammalian expression cassette for sgRNA, and lentiviral cis elements for packaging.

Notes

• Exact plasmid length, sequence map, promoters, selection markers, and spacer sequence are not specified for this item; refer to CoA/Spec Sheet.

Synthetic Utility

Applicability note: For a genetic construct, “synthetic utility” refers to molecular biology and genome engineering workflows rather than small‑molecule synthesis.

• Literature/general utility (not item-specific)

  • Modular cloning: The sgRNA spacer can be inserted via annealed oligos using Golden Gate (Type IIS) or Gibson/HiFi assembly. The plasmid backbone can be customized to swap promoters, selection markers, or add fluorescent reporters.
  • Genome engineering: When co‑expressed with a compatible Cas nuclease, the CDK5RAP2‑targeting sgRNA enables targeted DSBs. Pairing with donor templates supports HDR‑mediated edits (tags, point mutations) at the CDK5RAP2 locus.
  • Pooled screening: Lentiviral sgRNA backbones support pooled loss‑of‑function screens with NGS deconvolution (barcodes or guide sequences) to interrogate pathways tied to centrosome biology.
  • Multiguide strategies: Combine multiple sgRNAs in tandem expression cassettes for robust knockout or large deletions around CDK5RAP2 exons (backbone permitting).

• Practical considerations

  • Sequence verification of spacer and scaffold is essential to ensure on‑target performance.
  • Maintain supercoiled DNA for efficient transfection/packaging.
  • For HDR, use synchronized cells, short exposure to nuclease, and donor format (ssODN vs dsDNA) appropriate to edit size.

• Item-specific note

  • Exact cloning sites, antibiotic marker, promoter, and backbone features are not specified for this item; consult the CoA/Spec Sheet.

Target Specificity

• Product Data (item-specific)

  • The precise sgRNA spacer sequence, target exon, and PAM context for CDK5RAP2 are not specified for this item; refer to CoA/Spec Sheet.

• Literature/general guidance on sgRNA specificity (not item-specific)

  • PAM requirement: Ensure the sgRNA is designed adjacent to the PAM required by the chosen nuclease (e.g., NGG for SpCas9, TTTV for AsCas12a). Mismatches near the PAM‑proximal seed region disproportionately reduce on‑target activity.
  • Off‑target minimization: Use genome‑wide scoring tools (e.g., CFD/Rule Set 2 for SpCas9) to select guides with minimal predicted off‑targets; avoid guides with multiple perfect matches in the genome.
  • Genomic context: Favor constitutive exons common to all isoforms for knockout; target early coding exons to maximize frameshift likelihood. Check for SNPs in your cell line/organism that might affect binding.
  • Empirical confirmation: Validate on‑target cleavage and absence of high‑risk off‑targets via amplicon NGS, GUIDE‑seq/CIRCLE‑seq (as appropriate), and phenotype rescue with CRISPR‑resistant cDNA.
  • Multiplexing: Two independent guides against CDK5RAP2 increase confidence in phenotype attribution when both yield concordant results.

• Controls

  • Include non‑targeting and safe‑harbor targeting guides as negative/benchmark controls to interpret CDK5RAP2‑specific effects.

Frequently Asked Questions

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.

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-CDK5RAP2-sgRNA); cross-reactivity in untested species may exist but is not guaranteed. Contact technical support for specific species inquiries.