pLenti-UNK-sgRNA

Cat. No.: P751653

Specifications

Product Name

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

Application Protocols

Tested applications and recommended conditions: Not specified for this item; refer to CoA/Spec Sheet.

Note: In keeping with the requirement to only report protocols from product data, no procedural steps are provided here. Consult the product’s CoA/Spec Sheet or contact technical support for vector maps, cloning instructions, and application guidelines specific to this item.

Biological Roles

Item-specific biological function: Not specified for this item; refer to CoA/Spec Sheet.

General/literature context:

• sgRNA (single-guide RNA) is a synthetic fusion of CRISPR crRNA and tracrRNA that directs Cas nucleases to complementary DNA sequences via base pairing, enabling locus-specific double-strand breaks (e.g., with SpCas9) or binding (with dCas9).
• In lentiviral plasmid systems, the sgRNA cassette is typically transcribed by a Pol III promoter (often U6). Upon delivery to cells (transient transfection or lentiviral integration), the sgRNA associates with Cas protein supplied in trans or encoded by another vector.
• Biological consequences depend on the nuclease employed: Non-homologous end joining (NHEJ) commonly produces indels for gene knockout; homology-directed repair (HDR) can be leveraged with donor templates; catalytically dead Cas fusions enable transcriptional repression (CRISPRi) or activation (CRISPRa).
• Off-target considerations: Mismatches near the PAM-proximal seed region are least tolerated; genome- and cell type–specific chromatin states influence activity.

Important: This product is for research use only. No clinical, diagnostic, or therapeutic use is claimed or supported.

Buffer Applications

Item-specific buffer data: Not specified for this item; refer to CoA/Spec Sheet for any supplied buffer or recommended diluent.

General/literature guidance for plasmid DNA handling:

• Common storage buffers: TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) protects DNA by chelating Mg2+ required by nucleases. For enzyme reactions or transfections, nuclease-free water or low-salt Tris buffers (e.g., 10 mM Tris, pH 7.5–8.5) are often preferred.
• Working concentrations: Adjust with sterile, nuclease-free solutions; avoid repeated freeze–thaw.
• Electrophoresis buffers: TAE or TBE for agarose gel analysis of plasmid forms (supercoiled, nicked, linear). Use appropriate DNA stains; handle with care according to their safety profiles.
• Sterility: For cell culture applications, filter through 0.22 µm when compatible to reduce bioburden.

Note: If this product arrives lyophilized or at a defined concentration, reconstitution or dilution specifics should be obtained from the item’s CoA/Spec Sheet.

Green Alternatives

Context: This product is a biological DNA construct rather than an organic solvent or small-molecule reagent; green chemistry considerations focus on process choices rather than replacing the reagent itself.

Greener practice suggestions (literature/general):

• Minimize hazardous solvents: Prefer aqueous buffers for plasmid handling; avoid phenol-chloroform where column-based cleanups suffice.
• Solvent-free cloning: Use enzymatic assembly (Gibson/HiFi, Golden Gate) with minimal toxic reagents; reduce ethidium bromide use by switching to safer DNA stains.
• Waste reduction: Employ mini/midi preps sized to actual needs; pool colonies to reduce culture volumes when screening.
• Energy efficiency: Plan batch work to reduce freeze–thaw events and cold storage door-open time; consolidate shipments on dry ice where feasible.

Illustrative comparison (general):

• Phenol–chloroform extraction vs silica spin-column: Columns avoid halogenated waste, improve safety, and typically yield comparable purity for many workflows.
• Ethidium bromide vs SYBR Safe/ GelGreen: Safer alternatives reduce hazardous waste classification while maintaining sensitivity.

Note: No item-specific greener substitute exists for a particular plasmid sequence; sustainability gains come from workflow optimization.

Pharmaceutical Uses

Not typically applicable. This item is supplied for research use only and is not an excipient or GMP-grade material.

General/literature context:

• Plasmid DNA constructs can be used in discovery research, process development, and analytical method development, but they are not suitable for clinical manufacturing without appropriate GMP production, release testing, and regulatory documentation.
• When transitioning concepts toward regulated applications, key considerations include sequence fidelity, residual host impurities, endotoxin, bioburden, and compliance with pharmacopeial/ICH expectations. None of these attributes are specified for this item; refer to CoA/Spec Sheet.

No therapeutic or diagnostic claims are made for this product.

Physical Properties

Item-specific physical data: Not specified for this item; refer to CoA/Spec Sheet.

General/literature context for plasmid DNA materials:

• Aggregated thermophysical constants such as BP/MP, density, and refractive index are not typically defined for macromolecular DNA reagents.
• Solubility (literature): plasmid DNA is soluble in aqueous buffers (e.g., TE, Tris, nuclease-free water) and insoluble in most nonpolar organic solvents. Stability and apparent solubility depend on ionic strength and pH (commonly pH 7.5–8.5 for storage buffers).
• pKa/logP: Not applicable for high-molecular-weight polyanionic biopolymers like DNA.
• UV characteristics (literature): DNA exhibits a strong absorbance peak at ~260 nm (A260), with A260/A280 used to assess purity. Conversion (approximate): 1 A260 unit ≈ 50 µg/mL for dsDNA, though this depends on sequence and conformation.
• Viscosity/handling: Concentrated plasmid DNA solutions can be viscous; avoid vigorous vortexing to reduce shearing.

Note: For any item-specific values (concentration, buffer composition, endotoxin), consult the Certificate of Analysis (CoA)/Spec Sheet.

Quality and Grades

Item-specific quality/grade:

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

General guidance (literature/common practice for plasmid DNA reagents):

• Typical quality attributes include sequence verification (Sanger/NGS), restriction digest mapping, endotoxin level (e.g., research-grade vs. endotoxin-reduced for cell culture), OD260/280 purity, supercoiled percentage, and sterility testing.
• Research-grade plasmid DNA is suitable for standard molecular biology workflows (cloning, transfection). For sensitive applications (primary cells, in vivo research), low endotoxin preparations are often preferred; consult specification.
• UV absorbance metrics: A260/280 ~1.8–2.0 and A260/230 >2.0 are commonly used acceptance ranges (literature), but exact acceptance criteria are item- and supplier-specific.
• Stabilizers/preservatives: If present (e.g., EDTA in TE buffer), they chelate divalent cations to inhibit nucleases. For this item, any stabilizer or buffer composition is not specified; refer to CoA/Spec Sheet.

Recommendation: Verify lot-specific concentration, buffer, and QC attributes in the CoA prior to critical experiments.

Reaction and Applications

Item-specific applications: Not specified beyond the name; refer to CoA/Spec Sheet.

General/literature applications for pLenti sgRNA plasmids:

• CRISPR genome engineering: Expression of a single-guide RNA to direct Cas9/Cas12 nucleases to a target genomic locus. The “UNK” designation suggests a user-defined spacer sequence to be cloned/inserted.
• Lentiviral delivery workflows: Use with packaging plasmids to produce replication-incompetent lentiviral particles for stable expression of sgRNA in mammalian cells. Requires BSL-2 practices and institutional approvals.
• Cloning and screening: Spacer insertion via BsmBI/BbsI Golden Gate or Gibson Assembly (vector dependent), followed by colony PCR/Sanger sequencing to verify spacer identity.
• Pooled screening: Libraries of sgRNAs can be cloned into pLenti backbones for loss-of-function screens; downstream analysis involves NGS of integrated barcodes/sgRNA cassettes.
• Functional genomics: Knockout, CRISPRi (if dCas9-KRAB is co-expressed), or CRISPRa (if dCas9-activation domains are present) depending on the system into which the sgRNA is introduced.

Practical tips (literature):

• Maintain high supercoiled percentage for efficient transfection/transduction yield.
• Validate spacer on-target efficiency and off-target risk with computational tools; empirically test multiple candidates.
• For lentiviral use, confirm vector map (e.g., LTRs, Psi, promoter) and antibiotic marker; exact features for this item are not specified.

Reaction Conditions

Item-specific conditions: Not specified for this item; refer to CoA/Spec Sheet.

General/literature guidance for common workflows:

• Spacer insertion (Golden Gate): Typical reaction with BsmBI/BbsI and T4 ligase in a single-pot, thermocycling between 37 °C (digest) and 16 °C (ligate) for 10–30 cycles; total 0.5–2 h. Requires vector-specific overhangs.
• Gibson/HiFi assembly: 50 °C for 15–60 min with exonuclease/polymerase/ligase mix; design ~20–40 bp overlaps flanking the spacer region.
• Bacterial transformation: Chemically competent E. coli at 42 °C heat shock (30–60 s) or electrocompetent cells by electroporation; recover in SOC at 30–37 °C as per strain guidance; plate on the appropriate antibiotic.
• Lentiviral packaging (BSL-2; institutional approval required): Transfect HEK293T or similar producer cells with transfer vector + packaging plasmids; harvest supernatant 48–72 h. Optimize transfection reagent, DNA mass ratios, and culture conditions per lab SOP. This item’s packaging details are not specified.

All conditions above are general literature guidance and must be adapted to the specific vector map and institutional biosafety requirements.

Safety and Handling

Item-specific hazard/GHS data:

• Signal Word: Not specified for this item; refer to SDS.
• H-Statements: Not specified for this item; refer to SDS.
• GHS Classification and Pictograms: Not specified for this item; refer to SDS.

General laboratory safety (literature/practice for plasmid DNA/lentiviral backbones):

• Handle using good microbiological practices. Wear appropriate PPE: lab coat, gloves, and safety glasses. Work on clean, RNase/DNase-free surfaces to prevent degradation/contamination.
• Biological risk: Plasmid DNA itself is generally handled at BSL-1. However, lentiviral backbones are often used to produce viral particles; viral packaging and transduction activities should follow institutional biosafety approvals and appropriate containment (commonly BSL-2). Follow your IBC guidance.
• Incompatibilities: Avoid nucleases (RNase/DNase) and strong oxidants; avoid repeated freeze–thaw cycles that can shear DNA.
• First aid (general): In case of contact with eyes/skin, rinse with water. If inhaled (aerosolized dust from lyophilizates), move to fresh air. If ingested, rinse mouth. Seek medical attention if symptoms persist. Defer to SDS for authoritative guidance.
• Waste: Dispose of DNA-contaminated materials per institutional biological waste procedures.
• Transport: Keep cold to preserve integrity; avoid uncontrolled warming during transit or handling.

Solvent Selection

Applicability: This product is a plasmid/sgRNA construct, not a small-molecule solute; classical solvent selection paradigms (polarity, dielectric constant) are not directly applicable.

General/literature guidance:

• Working medium: Aqueous buffers are standard (e.g., TE: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) or nuclease-free water. Avoid divalent cations (Mg2+, Ca2+) unless required for downstream enzymes.
• Incompatibility: Poorly soluble/stable in most organic solvents; exposure can precipitate or denature DNA.
• Practical choices:
  • For long-term storage: TE buffer helps protect against nuclease activity (EDTA chelation).
  • For transfection or enzyme reactions: Follow kit-specific buffer requirements; many transfection reagents prefer nuclease-free water or low-salt Tris.
• If concentration is needed: Use ethanol/isopropanol precipitation or spin columns; avoid organic cosolvents in final preparations for biological assays.

Storage and Reconstitution

Item-specific storage and shipping (from Product Data):

• Storage Conditions: Store at -80 °C.
• Shipped In: Dry ice packs + Cold packs.

General/literature guidance:

• Minimize freeze–thaw cycles by aliquoting upon first receipt. Keep samples in low-DNA-binding tubes. Avoid repeated warming above -20 °C.
• If lyophilized: Reconstitute with sterile, nuclease-free water or TE buffer to the desired concentration; mix gently by pipetting (avoid vigorous vortexing). Exact reconstitution volume or target concentration is not specified for this item; refer to CoA/Spec Sheet.
• If supplied in solution: Note the buffer composition and DNA concentration from the CoA; store at -80 °C as specified. For short-term use, -20 °C storage may be acceptable in many labs, but adhere to item-specific guidance.
• Handling: Use RNase/DNase-free techniques and consumables. For cell-based work, maintain aseptic technique and consider 0.22 µm filtration if compatible.

For authoritative instructions on buffer composition, concentration, and stability claims, consult the lot-specific CoA/Spec Sheet.

Structure and Identity

Item-specific facts (from Product Data):

• SKU: P751653
• Product Name: pLenti-UNK-sgRNA
• CAS: Not specified for this item; refer to CoA/Spec Sheet.
• CID: Not specified for this item; refer to CoA/Spec Sheet.
• SMILES: Not specified for this item; refer to CoA/Spec Sheet.
• InChIKey: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular Formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular Weight: Not specified for this item; refer to CoA/Spec Sheet.

General/literature context (for plasmid DNA reagents):

• pLenti designations typically refer to lentiviral plasmid backbones engineered for packaging into replication-incompetent lentiviral vectors.
• “sgRNA” denotes a single-guide RNA expression cassette (commonly under U6 or H1 promoters) for CRISPR applications. “UNK” implies an unspecified or user-defined spacer/protospacer sequence placeholder.
• Molecular structure: plasmids are circular, double-stranded DNA molecules (supercoiled/nicked forms possible), not well-described by line notations like SMILES; identity is defined by nucleotide sequence, map, and features (promoters, selection markers, LTRs, packaging signals).
• Structural features (typical, literature): LTRs/5' and 3', Psi packaging signal, promoter(s) for sgRNA, bacterial origin of replication, and antibiotic resistance marker for selection. Exact elements for this item are not specified.

Synthetic Utility

Chemical synthesis context: Not applicable as a classical small-molecule building block.

Molecular biology “synthetic” utility (literature/general for sgRNA plasmids):

• Vector as a chassis: Serves as a backbone for inserting user-defined sgRNA spacers via Golden Gate (type IIS restriction enzymes), Gibson/HiFi assembly, or ligation cloning.
• Modular engineering: Facilitates rapid construction of CRISPR toolsets (e.g., swapping promoters, adding barcodes, integrating reporters or selection markers) using standard BioBrick/Golden Gate/Gibson strategies.
• Library synthesis: Supports pooled oligo library cloning for genome-scale screens; compatible with NGS-based readout of sgRNA representation.
• Downstream utility: Enables generation of stable cell lines (via lentiviral delivery) to couple with Cas9/Cas12 expression systems for knockout, CRISPRi, or CRISPRa studies.

Note: Exact restriction sites, overhangs, and vector map for this specific item are not provided; consult the CoA/Spec Sheet for authoritative cloning details.

Target Specificity

Item-specific target details: Not specified for this item; refer to CoA/Spec Sheet.

General/literature note:

• sgRNA specificity is determined by the spacer sequence and the PAM requirement of the chosen Cas enzyme (e.g., 5'-NGG-3' PAM for SpCas9). As the spacer for “UNK” is unspecified, no target information can be provided for this product listing.

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