pLenti-TKFC-sgRNA

Cat. No.: P751094

Specifications

Product Name

pLenti-TKFC-sgRNA

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

The following protocols are general, literature‑based guidance for pLenti sgRNA vectors and are not product‑specific specifications.

• A. Plasmid propagation

  • Transform into high‑efficiency E. coli (e.g., Stbl3 for lentiviral backbones). Plate on appropriate antibiotic (marker not specified for this item). Culture at 30–37°C; avoid overgrowth to limit recombination. Prepare endotoxin‑reduced plasmid DNA.

• B. Guide insertion (if vector is a cloning scaffold)

  • Anneal oligos with overhangs matching Type IIS sites (e.g., BsmBI) and ligate using Golden Gate. Verify insertion by colony PCR and Sanger sequencing.

• C. Transfection (target cells or HEK293T for packaging)

  • Mix plasmid DNA with lipid/polymer reagent per manufacturer. Incubate 10–20 min, add to cells at ~50–80% confluence. Replace medium after 4–24 h.

• D. Lentiviral packaging and transduction

  • Co‑transfect transfer vector with packaging plasmids in HEK293T. Collect supernatants at 48/72 h, filter (0.45 µm), and transduce target cells with or without polybrene (4–8 µg/mL). Select with the vector’s antibiotic (if present; not specified here).

• E. Editing validation

  • Extract genomic DNA 48–96 h post‑delivery. PCR‑amplify the TKFC locus; assess editing by T7E1, ICE/TIDE, or amplicon NGS. Confirm functional impact by qPCR/Western as appropriate.

Note: Exact antibiotic marker, cloning sites, and insert sequence for pLenti‑TKFC‑sgRNA are not specified; refer to CoA/Spec Sheet.

Biological Roles

• Target gene context (literature; not product specification)

  • TKFC (Triokinase/FMN cyclase) encodes a bifunctional enzyme with two activities: (1) triokinase activity, phosphorylating triose sugars such as dihydroxyacetone and glyceraldehyde, thereby linking to glycolytic and fructose metabolism; and (2) FMN cyclase activity, converting FMN to cyclic FMN. TKFC is localized primarily in the cytosol and is expressed in multiple tissues.
  • Pathway relevance: TKFC participates in fructose and glycerol/triose metabolism, influencing cellular energy balance and carbon flux into glycolysis. The FMN cyclase function impacts flavin nucleotide homeostasis.
  • Genetic perturbation outcomes (reported in literature): Loss‑of‑function can alter triose phosphate pools and downstream metabolic flux; overexpression or suppression may change redox‑associated pathways due to flavin handling. Specific phenotypes are cell‑type dependent.

• How an sgRNA construct interfaces with biology (general)

  • CRISPR/Cas9 editing: sgRNA directs Cas9 to a genomic locus adjacent to a PAM sequence (e.g., NGG for SpCas9). Double‑strand breaks typically repair via NHEJ, creating indels that can disrupt the TKFC reading frame.
  • CRISPRi/a modulation: sgRNA recruits catalytically dead Cas9 fusions to promoter or proximal regions to sterically inhibit or recruit transcriptional machinery, respectively, modulating TKFC expression without altering DNA sequence.

Note: The specific guide target site(s), PAM, and on/off‑target characteristics for pLenti‑TKFC‑sgRNA are not specified for this item; consult the CoA/Spec Sheet.

Buffer Applications

This product is a plasmid DNA reagent; it is not a classical buffering agent. However, handling and storage buffers are critical to performance.

• Item-specific (from Product Data)

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

• Practical buffer systems for plasmid DNA (general guidance)

  • TE buffer (10 mM Tris‑HCl, 1 mM EDTA, pH 8.0): Preferred for long‑term storage; EDTA chelates Mg2+ and inhibits DNases.
  • Low‑EDTA TE (10 mM Tris‑HCl, 0.1 mM EDTA, pH 8.0): Balances stability with compatibility for downstream enzymatic reactions.
  • Nuclease‑free water (pH ~7.0–8.0): Suitable for PCR and short‑term storage; consider aliquoting for −80°C storage to reduce freeze‑thaw.

• Recipe hints (literature)

  • Tris‑HCl 1 M stock (pH 8.0); combine to make 10 mM final, add EDTA to desired concentration, filter sterilize (0.22 µm), and store at room temperature.
  • Avoid divalent cations (Mg2+, Ca2+) in storage buffers; they promote nuclease activity and DNA degradation.

• Electrophoresis/context use

  • For plasmid QC, use TAE or TBE running buffers. Note that EDTA in samples can slightly affect migration; maintain consistent conditions across samples.

Green Alternatives

While this is not a chemical solvent, sustainability can be improved in workflows using plasmid DNA and lentiviral systems.

• Opportunities (general, literature)

  • Delivery modality: Consider Cas9 RNP (ribonucleoprotein) delivery with synthetic sgRNA to avoid viral packaging and associated biowaste, when transient editing suffices.
  • Non‑viral vectors: Use non‑viral episomal or transposon systems (e.g., piggyBac, Sleeping Beauty) to reduce BSL‑2 operations and bleach/chemical disinfectant use; weigh integration profiles and stability.
  • Mini/prep methods: Choose endotoxin‑free, alcohol‑sparing plasmid prep kits and recycle ethanol/IPA where feasible. Implement vacuum manifolds to lower energy use versus high‑speed centrifugation.
  • Media/reagents: Opt for animal‑component‑free media and biodegradable plastics where performance allows; consolidate single‑use plastics to reduce waste.

• Trade‑offs

  • RNP delivery reduces off‑target window and viral waste but may have lower efficiency in hard‑to‑transfect lines and offers limited stable expression.
  • Non‑viral systems avoid viral steps but differ in cargo capacity, integration preferences, and selection requirements.

• Process choices that reduce hazards

  • Use neutral pH DNA storage buffers instead of high‑salt/chaotropic residues.
  • Implement closed‑system filtration for buffer exchange to minimize solvent exposure and spills.

Pharmaceutical Uses

Not applicable. This product is supplied for research use only and is not intended for human or veterinary use, diagnostics, or as a pharmaceutical ingredient.

• General context (for process development research only; not product specification)
  • Lentiviral sgRNA vectors are used in discovery‑phase research to generate edited or modulated cell lines for target validation, mechanism studies, and screening campaigns.
  • In translational research settings (non‑clinical), similar constructs may inform manufacturability or process parameters; however, this listing does not specify GMP status, release testing, or pharmacopeial compliance.

Always refer to institutional and regulatory guidelines. No therapeutic, diagnostic, or clinical claims are made for this item.

Physical Properties

• Item-specific (from Product Data)

  • Appearance/concentration: Not specified for this item; refer to CoA/Spec Sheet.
  • Molecular weight/length: Not specified for this item; refer to CoA/Spec Sheet.

• General/literature values for plasmid DNA (for context; not product specifications)

  • Physical state: Aqueous buffered solution (e.g., TE or nuclease‑free water) or lyophilized DNA; colorless.
  • Solubility: Highly water-soluble; insoluble in organic solvents.
  • UV characteristics: Peak absorbance at 260 nm; A260 of 1.0 ≈ 50 µg/mL for double‑stranded DNA (literature).
  • A260/A280 ratio: ~1.8 indicates low protein contamination (literature guidance).
  • Storage buffer: Frequently TE (10 mM Tris‑HCl, 1 mM EDTA, pH 8.0) or nuclease‑free water; exact buffer for this item not specified.
  • Thermal behavior: DNA denatures on heating (>90°C) and is susceptible to nuclease degradation; freeze‑thaw cycles can shear DNA.

• Not applicable properties for this biomaterial

  • Classical small‑molecule constants (bp, mp, density, logP, refractive index, SMILES, InChIKey) do not apply to plasmid DNA constructs.

Note: For exact concentration, buffer composition, and plasmid length for pLenti‑TKFC‑sgRNA, see the item’s CoA/Spec Sheet.

Quality and Grades

• Item-specific (from Product Data)

  • Grade/purity: Not specified for this item; refer to CoA/Spec Sheet.
  • Appearance, concentration, endotoxin level, sequence verification: Not specified for this item; refer to CoA/Spec Sheet.

• How to interpret typical quality attributes for plasmid/viral vectors (general guidance)

  • Sequence identity: For sgRNA vectors, correct guide sequence and backbone junctions are commonly verified by Sanger sequencing. Full plasmid NGS is increasingly used.
  • Purity: Assessed by A260/A280 and A260/A230 ratios; agarose gel to confirm supercoiled predominance. Endotoxin level (EU/µg DNA) is critical for cell culture and in vitro transfection.
  • Functional QC: Test transfection into HEK293T and/or cloning enzyme digestion patterns to confirm vector integrity. For lentiviral use, titering of packaged virus (TU/mL) validates performance but is not a property of the plasmid itself.
  • Stabilizers: DNA is often supplied in TE buffer; EDTA helps chelate divalent cations and suppress nuclease activity. Presence/absence for this item is not specified.
  • Grades you may encounter: “Research grade” (typical for lab use), “Endotoxin‑reduced/Transfection‑grade,” or “GMP‑like” for process development. This listing does not specify a grade.

Please consult the CoA/Spec Sheet for exact QC metrics and acceptance criteria specific to pLenti‑TKFC‑sgRNA.

Reaction and Applications

• Item-specific (from Product Data)

  • Intended use: Research use only. Specific application details not provided; refer to CoA/Spec Sheet.

• General applications for pLenti sgRNA constructs (literature/practice)

  • CRISPR/Cas9 gene knockout: Co‑deliver Cas9 and this sgRNA vector to disrupt the TKFC coding sequence via NHEJ. Use T7E1, ICE/TIDE, or amplicon NGS to quantify indels.
  • CRISPRi/CRISPRa: When paired with dCas9‑KRAB or dCas9‑VP64/CRISPRa systems, sgRNA guides can repress or activate TKFC transcription without genomic cuts.
  • Stable cell line generation: Package the sgRNA cassette into pseudotyped lentiviral particles using 2nd/3rd‑generation systems and transduce target cells; select with the vector’s resistance marker (if present; not specified here).
  • Multiplex editing: Clone additional sgRNAs using Golden Gate or Gibson assembly to target multiple TKFC sites or include non‑overlapping controls.

• Practical tips (general)

  • Guide design: Use on‑target and off‑target scoring tools; favor NGG‑adjacent protospacers if using SpCas9. Validate with at least two independent guides targeting TKFC.
  • Packaging: Maintain high plasmid purity and low endotoxin; typical transfection into HEK293T at DNA ratios following the packaging system vendor guidance.
  • Titration: Determine functional titer (TU/mL) via antibiotic selection or qPCR‑based methods prior to large‑scale experiments.
  • Controls: Include non‑targeting and safe‑harbor locus guides; verify TKFC perturbation at mRNA (RT‑qPCR) and protein (western blot) levels.

All procedure parameters above are general literature guidance and not specifications for this item.

Reaction Conditions

The plasmid itself is not a reagent in a chemical reaction; however, typical biological “reaction” conditions apply for transfection, lentiviral packaging, and transduction. The following are general literature guidelines and not product specifications.

• Transfection (HEK293T for packaging or target cells)

  • DNA purity: Endotoxin‑reduced; A260/280 ~1.8–2.0.
  • Complexation: Follow vendor ratios for lipid/polymer reagents; commonly 1–3 µg DNA per well (6‑well) with serum‑free medium during complexation.
  • Conditions: 37°C, 5% CO2; change to fresh complete medium after 4–24 h depending on reagent.

• Lentiviral packaging (3rd‑generation system)

  • Plasmid ratios (mass): Transfer:pMDLg/pRRE:RSV‑Rev:VSV‑G often ~4:3:2:1 (optimize per system). Harvest supernatant at 48 and 72 h.
  • Filtration: 0.45 µm PES; optional concentration by PEG precipitation or ultracentrifugation.

• Transduction

  • MOI: Start with MOI 0.3–5 depending on cell type and selection strategy.
  • Polybrene: 4–8 µg/mL can enhance entry (check cell tolerance).
  • Selection: Begin 24–72 h post‑transduction using the vector’s antibiotic (if present; not specified here); determine minimal lethal dose beforehand.

• Editing assessment

  • Timeline: Peak indel formation 48–96 h post Cas9+sgRNA delivery. Validate on‑target edits by amplicon sequencing and off‑target with GUIDE‑seq/CIRCLE‑seq as needed.

Safety and Handling

• Item-specific (from Product Data)

  • GHS classification, 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 biosafety guidance for lentiviral plasmids and sgRNA constructs (literature/practice; not product specifications)

  • Biosafety level: Plasmid DNA alone can be handled at BSL‑1. However, production of replication‑incompetent lentiviral particles typically requires BSL‑2 containment and institutional approvals.
  • PPE: Lab coat, gloves (nuclease‑free best practice), and eye protection. Change gloves frequently to prevent nuclease contamination.
  • Engineering controls: Use a certified biosafety cabinet when handling viral packaging mixes, transductions, or potentially infectious supernatants.
  • Incompatibilities: Avoid nucleases (DNase), microbial contamination, and repeated freeze‑thaw. Keep solutions nuclease‑free; clean work surfaces with DNA‑degrading agents where appropriate.
  • Spill/Exposure (plasmid solutions): Wipe with 10% bleach followed by water; for viral supernatant spills, use BSL‑2 procedures per institutional biosafety manual.
  • First aid (general): If contact with eyes/skin occurs, rinse with water. If inhaled/ingested, seek medical attention as per SDS.

Always consult the product’s SDS and your Institutional Biosafety Committee (IBC) for authoritative safety and compliance requirements.

Solvent Selection

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

• Item-specific (from Product Data)

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

• Practical buffer choices for handling plasmid DNA (general guidance)

  • Nuclease‑free water: Suitable for short‑term use, PCR, cloning, and in vitro transcription; consider aliquoting and storing at −80°C to minimize freeze‑thaw.
  • TE buffer (10 mM Tris‑HCl, 1 mM EDTA, pH 8.0): Promotes long‑term DNA stability by chelating Mg2+ and inhibiting nucleases; may be suboptimal for some enzyme reactions sensitive to EDTA.
  • Low‑EDTA Tris buffer (10 mM Tris‑HCl, 0.1 mM EDTA): Balance between stability and downstream enzymatic compatibility.
  • For transfection: Many protocols dilute DNA in Opti‑MEM or similar reduced‑serum medium immediately before complexation with lipids or polymers; follow reagent‑specific recommendations.

• Incompatibilities (general)

  • Avoid phenol, chloroform, alcohol carryover, and high salt in final preparations destined for transfection or viral packaging, as these can reduce cell viability and transfection efficiency.

Storage and Reconstitution

• Item-specific (from Product Data)

  • Storage conditions: Store at −80°C.
  • Shipping: Dry ice packs + Cold packs.

• Additional handling guidance (general; not product specification)

  • Upon receipt: Keep frozen. Minimize time at ambient temperature.
  • Aliquoting: Thaw on ice, gently mix by flicking or slow pipetting. Aliquot into nuclease‑free tubes to avoid repeated freeze‑thaw cycles that can shear DNA.
  • Working stocks: Store short‑term (days to weeks) at −20°C; long‑term at −80°C. Avoid more than 3–5 freeze‑thaw cycles.
  • Buffer: If supplied in nuclease‑free water, consider adjusting to TE (10 mM Tris‑HCl, 0.1–1 mM EDTA, pH 8.0) for long‑term stability unless EDTA interferes with your downstream application. The supplied buffer for this item is not specified.
  • Reconstitution (if lyophilized): Rehydrate with nuclease‑free water or TE to desired concentration (e.g., 0.1–1.0 µg/µL), incubate on ice 10–15 min, and mix gently until fully dissolved. Lyophilization status for this item is not specified.
  • Documentation: Record thaw cycles, concentrations (A260), and buffer composition on the vial and in LIMS.

For precise concentration, buffer composition, and any stabilizers used in pLenti‑TKFC‑sgRNA, consult the CoA/Spec Sheet.

Structure and Identity

Brief description: pLenti-TKFC-sgRNA is a lentiviral plasmid construct designed to express a CRISPR single-guide RNA (sgRNA) targeting the human TKFC gene (Triokinase/FMN cyclase).

• Item-specific (from Product Data)

  • Product type: Lentiviral sgRNA expression vector (name implies pLenti backbone with sgRNA against TKFC)
  • Sequence/insert details: Not specified for this item; refer to CoA/Spec Sheet.
  • Molecular formula: Not applicable for plasmid DNA; not specified for this item; refer to CoA/Spec Sheet.
  • Molecular weight/size (bp): Not specified for this item; refer to CoA/Spec Sheet.

• General/literature description (not item-specific)

  • Backbone: pLenti-class vectors typically include 5′ and 3′ LTRs, ψ packaging signal, RRE, cPPT/CTS, and bacterial origin of replication with antibiotic resistance marker for E. coli propagation.
  • sgRNA expression: Commonly under U6 or H1 RNA polymerase III promoter with a standard tracrRNA scaffold for Cas9 loading.
  • Optional cassettes: Many pLenti sgRNA vectors contain a selectable marker (e.g., puromycin, blasticidin) and/or a fluorescent reporter; presence/identity for this item is not specified.

• Structural features (2D/functional elements, literature)

  • Functional modules: LTRs (integration), packaging signal (ψ), promoter(s), sgRNA scaffold, bacterial Ori/selection marker.
  • Topology: Circular double‑stranded DNA plasmid in E. coli; integrates only when packaged into pseudotyped lentiviral particles and delivered to mammalian cells with functional integrase.

• Registry identifiers (item-specific)

  • CAS, CID, InChIKey, SMILES: Not applicable/not specified for plasmid DNA constructs; refer to CoA/Spec Sheet.

Synthetic Utility

Classical small‑molecule “synthetic utility” does not apply to a plasmid DNA construct. Instead, consider its utility in molecular biology and genetic engineering workflows.

• Functional modules enabling cloning (general)

  • Multiple cloning sites and Type IIS sites (e.g., BsmBI/BbsI) in many sgRNA vectors allow rapid insertion/replacement of protospacer sequences using Golden Gate assembly; presence for this item is not specified.
  • Bacterial propagation and selection (e.g., AmpR, KanR) permit high‑yield plasmid preparation; the specific marker for this item is not specified.

• Retrosynthetic perspective (vector engineering)

  • Modular design: sgRNA cassette, promoter, selectable marker, and lentiviral elements can be swapped or combined to create new constructs (e.g., dual‑sgRNA arrays, inducible promoters).
  • Accessory elements: cPPT/CTS and WPRE elements (if present) can enhance transduction and expression; inclusion for this item is not specified.

• Downstream use

  • As a donor for Gibson/HiFi assembly to build TKFC perturbation toolsets (knockout, CRISPRi/a, base/prime editing when paired with appropriate effectors).

For exact restriction map, antibiotic selection, and cloning sites of pLenti‑TKFC‑sgRNA, consult the CoA/Spec Sheet.

Target Specificity

• Item-specific (from Product Data)

  • Target: TKFC gene implied by product name.
  • Guide sequence, exon/intron position, strand, PAM context, and off‑target analysis: Not specified for this item; refer to CoA/Spec Sheet.

• General considerations for sgRNA specificity (literature)

  • On‑target efficacy depends on protospacer sequence composition, chromatin accessibility, and proximity to functional domains (e.g., early coding exons for knockout).
  • Off‑target risk increases with sequence homology at seed regions (proximal to PAM). Use in silico tools to predict and rank potential off‑targets and avoid sites with high similarity in coding regions.
  • PAM requirements: For SpCas9, NGG is canonical; alternative nucleases (SaCas9, Cas12a) have distinct PAMs and spacer lengths. This item does not specify the associated nuclease.
  • Validation: Employ at least two independent sgRNAs targeting TKFC and include non‑targeting controls. Confirm gene perturbation by both genotypic and phenotypic assays.

Consult the CoA/Spec Sheet for the exact target protospacer and any provided specificity metrics.