pLenti-B2M-sgRNA

Cat. No.: P744046

Specifications

Product Name

pLenti-B2M-sgRNA

Product Line

Produits biologiques

Stockage et expédition

Conditions de stockage de stockage

Conserver à -80°C

Expédié en

Dry ice packs + Cold packs

Stabilité et stockage

Stocké à -20 ℃, valable pendant 3 mois ; Stocké à -80 ℃, valable pendant au moins un an.

Application Protocols

Item-specific, validated applications and dilutions: Not specified for this item; refer to CoA/Spec Sheet.

General protocols (literature/typical)

1. Lentiviral packaging and transduction

• Day 0: Seed HEK293T cells at 5–7 × 10^5 per well (6-well) to reach ~70% confluence next day.
• Day 1: Transfect with transfer plasmid (this sgRNA vector) + packaging plasmids using a lipid reagent in Opti-MEM. Replace medium after 6–12 h.
• Day 2/3: Harvest supernatant at 48/72 h, filter (0.45 µm). Optional: Concentrate virus. Determine titer (p24, RT, or functional titration).
• Transduction: Apply virus to target cells at desired MOI with polybrene (if compatible). Replace medium after 12–24 h.
• Selection: Start selection 48–72 h post-transduction; maintain until non-transduced controls are fully dead.

2. Direct plasmid transfection

• Plate target cells to 60–80% confluence. Mix plasmid with transfection reagent per vendor ratios. Incubate complexes 10–20 min, add to cells. Analyze/editing 48–96 h later.

3. Editing verification

• Flow cytometry: Stain for surface MHC I (e.g., HLA-ABC) to infer B2M disruption.
• Genotyping: PCR amplify target locus; perform Sanger + ICE/TIDE or amplicon NGS for indel quantification. Note: Adjust DNA amounts and reagent ratios per cell type and platform.

Biological Roles

General background on B2M (literature)

• Gene/protein: Beta-2-microglobulin (B2M) encodes a small secreted component that noncovalently associates with MHC class I heavy chains, essential for stable MHC I surface expression on nearly all nucleated cells.
• Cellular functions: Proper B2M is required for peptide loading and trafficking of MHC I to the plasma membrane, influencing antigen presentation to CD8+ T cells and shaping immune surveillance and NK cell interactions.
• Consequences of gene disruption (research context): Knockout of B2M typically reduces/abolishes MHC I on the cell surface, frequently used to create MHC I–null models, to benchmark CRISPR efficacy, or to modulate immunological assays.
• Redundancy and context: B2M function is unique within the MHC I complex; however, overall antigen presentation also depends on TAP1/2, tapasin, ERAP1/2, and proteasome activity. Note: The above summarizes biological context of the target gene. It does not describe the specific sgRNA sequence or performance of this item, which are not specified.

Buffer Applications

Not a classical buffer reagent. However, buffer choice impacts plasmid stability and performance (literature/typical):

• Storage: TE buffer (10 mM Tris-HCl, 0.1–1 mM EDTA, pH 7.5–8.5) protects against nuclease degradation; EDTA may inhibit downstream enzymes if not diluted.
• Working solutions: Nuclease-free water or low-salt Tris (10 mM) for transfection to reduce cytotoxicity and salt carryover.
• Viral packaging: Prepare transfection mixes in serum-free Opti-MEM or equivalent per transfection reagent guidance; return to complete medium after complex formation.
• Electroporation: Use manufacturer-recommended electroporation buffers; high-conductivity buffers can cause arcing. Recipes and exact conditions depend on cell type and transfection platform.

Green Alternatives

While the product is a biological plasmid reagent, greener practices can be adopted in its use and supporting workflows (literature/typical):

• DNA staining: Replace ethidium bromide with safer fluorescent dyes (e.g., SYBR Safe) for gel analysis to reduce mutagenic waste.
• Solvent minimization: Favor column-based plasmid purification over phenol–chloroform extractions; if solvents are necessary, ensure proper capture and disposal.
• Energy efficiency: Consolidate freezer storage and use high-efficiency −80°C freezers; minimize door openings and use inventory systems to reduce search time.
• Waste reduction: Use reusable ice packs, minimize single-use plastics where sterility allows, and adopt refillable pipette tip systems.
• Transfection: Prefer high-efficiency, serum-compatible reagents that allow lower DNA doses, reducing upstream culture and consumable use.
• Packaging scale: Right-size lentiviral production batches to experimental need to limit reagent and consumable waste. Tradeoffs: Some greener alternatives may have higher upfront cost or require validation to match performance.

Pharmaceutical Uses

Not applicable as a finished pharmaceutical or excipient. This plasmid DNA reagent is for research use only, not for diagnostic, therapeutic, or in vivo clinical applications.

General manufacturing context (literature/typical)

• Process development: Similar constructs may be used in preclinical R&D to study gene function in cell lines. Any GMP or clinical manufacturing would require distinct, qualified production, extensive characterization, and regulatory oversight—outside the scope of this item.

Physical Properties

Item-specific facts (from Product Data)

• Appearance, concentration, A260/280, endotoxin: Not specified for this item; refer to CoA/Spec Sheet.

General/literature values and notes for plasmid DNA reagents

• State: Circular double-stranded DNA, typically provided in TE or water. No meaningful boiling/melting point akin to small molecules; DNA denaturation temperature depends on sequence/conditions.
• Solubility: Highly water-soluble; stable in low-ionic-strength aqueous buffers (e.g., 10 mM Tris, 0.1–1 mM EDTA, pH 7.5–8.5) (literature).
• pKa/logP/refractive index: Not applicable to macromolecular DNA.
• Concentration units: Often supplied at 0.1–1.0 mg/mL or as lyophilized mass (literature); actual for this item not specified.
• Plasmid size: Typical lentiviral backbones are ~8–12 kb (literature); actual size for this item not specified.
• Optical properties: Quantified by A260 (1 A260 ≈ 50 µg/mL for dsDNA; literature). Exact absorbance ratios for this item are not specified.

Quality and Grades

Item-specific facts (from Product Data)

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

General guidance on quality attributes for plasmid DNA (literature/typical)

• Identity: Verified by Sanger sequencing across the sgRNA insert and junctions; full plasmid mapping by restriction analysis or NGS for critical applications.
• Purity: Monitored by A260/280 (~1.8–2.0) and A260/230 (>2.0) ratios; agarose gel electrophoresis to assess supercoiled vs nicked/linear forms.
• Endotoxin: Low-endotoxin preparations (<0.1 EU/µg) are preferred for mammalian transfection and lentiviral packaging to maximize titer and cell viability.
• Residual contaminants: Evaluate for RNase, DNase, phenol/solvent carryover, and salt content; these impact transfection efficiency.
• Stabilizers: Some preparations include EDTA in TE to chelate divalent cations and protect from nucleases; confirm compatibility with downstream enzymology.
• Documentation: CoA typically includes vector map, insert sequence, concentration, and QC metrics. For this item, consult the CoA/Spec Sheet for definitive specifications.

Reaction and Applications

Item-specific facts (from Product Data)

• Intended use: Research use only. No further application data specified for this item.

General applications for pLenti sgRNA constructs (literature/typical)

• Gene knockout/knockdown: Express sgRNA to direct Cas9-mediated indels at the B2M locus, commonly used to disrupt MHC I surface expression in human cells for functional genomics and immunology workflows.
• Stable cell line generation: Package into lentiviral particles and transduce target cells for durable sgRNA expression; select with the plasmid’s resistance marker (exact marker for this item not specified).
• Pooled CRISPR screening: Use as a single-guide positive control/benchmark in screens monitoring antigen presentation pathways.
• Complementary systems: Combine with Cas9-expressing cells or co-deliver Cas9 by plasmid, virus, mRNA, or protein. For CRISPRi/a, pair with dCas9-KRAB or dCas9-VP64 systems; note that an sgRNA optimized for knockout may require different design for CRISPRi/a.
• Practical tips: Maintain the sgRNA region during cloning/propagation in recombination-deficient E. coli strains; verify sequence before critical experiments. For lentiviral work, optimize MOI (e.g., ≤0.3 for single-copy integration in screens; literature) and include polybrene only if compatible with the cell type.

Reaction Conditions

Item-specific reaction/assay conditions are not provided. The following are general, literature-based conditions for typical use of pLenti sgRNA plasmids:

• Plasmid transfection (non-viral): 0.5–2 µg DNA per well (6-well format) with lipid-based reagent; incubate 24–72 h before analysis. Optimize DNA:reagent ratios per supplier instructions.
• Lentiviral packaging: Co-transfect HEK293T cells at ~70–80% confluence with transfer plasmid + packaging plasmids (e.g., gag/pol and VSV-G). Collect supernatants at 48 and 72 h; filter (0.45 µm). Concentrate if needed (PEG or ultracentrifugation). Work under BSL-2.
• Transduction: Aim for MOI ≤0.3 for single-copy integration in screens; include 4–8 µg/mL polybrene if cell-type compatible. Spinoculation (800–1200 × g, 60–90 min) may enhance uptake for refractory cells.
• Selection: Begin antibiotic selection 48–72 h post-transduction based on kill-curve results for your cell line and the plasmid’s resistance marker (not specified here).
• Editing assessment: Evaluate 3–7 days post-transduction by flow cytometry for MHC I, or via T7E1/ICE/amplicon NGS for indels. For clonal lines, single-cell sort 5–10 days post-selection. Note: Actual plasmid features and recommended conditions for this SKU are not specified; consult CoA/Spec Sheet and optimize empirically.

Safety and Handling

Item-specific facts (from Product Data)

• GHS classification, signal word, hazard statements, pictograms: Not specified for this item; refer to SDS.
• Storage condition: Store at −80°C (provided).

General biosafety guidance (literature/typical for lentiviral sgRNA plasmids)

• Biosafety level: Handle lentiviral vectors and packaging workflows in BSL-2 facilities with institutional approval and appropriate training.
• PPE: Lab coat, gloves, and eye protection. Use biological safety cabinet (Class II) for aerosol-generating steps (pipetting, transfection mixtures, harvesting supernatants).
• Incompatibilities: Avoid nucleases (DNases), repeated freeze–thaw cycles, and microbial contamination. Use nuclease-free reagents and plasticware.
• Viral work: Decontaminate surfaces with fresh 10% bleach followed by 70% ethanol. Collect liquid waste with bleach inactivation prior to disposal per local guidelines.
• First aid (overview): Skin/eye contact with plasmid solutions—rinse with water. Inhalation of aerosols during viral work—move to fresh air, seek medical evaluation per institutional exposure protocol. Always defer to site-specific SOPs and SDS.
• Transport: Maintain cold chain. Avoid dry ice entrapment of vials; use secondary containment. Note: For authoritative safety information, consult the product SDS and your institutional biosafety office.

Solvent Selection

Not typically applicable. This is an aqueous plasmid DNA reagent rather than a small-molecule solute requiring organic solvent optimization.

General notes (literature/typical)

• Solvent system: Use nuclease-free water or TE buffer (10 mM Tris-HCl, 0.1–1 mM EDTA, pH 7.5–8.5) for storage and gentle handling.
• Compatibility: Avoid strong acids/bases and organic solvents that denature DNA or interfere with transfection/packaging. For precipitation or cleanup, ethanol/isopropanol can be used in standard DNA protocols; thoroughly remove traces before cell-based use.
• Sterility: For cell culture applications, maintain sterility and use 0.22 µm filtration where appropriate.

Storage and Reconstitution

Item-specific facts (from Product Data)

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

General guidance for plasmid DNA (literature/typical)

• Short-term storage: For frequent use, aliquot and keep at −20°C to minimize freeze–thaw; avoid more than 3–5 cycles. For long-term archival, maintain at −80°C.
• Buffer: Store in nuclease-free TE (10 mM Tris-HCl, 0.1–1 mM EDTA, pH 7.5–8.5). For immediate transfection, DNA in nuclease-free water is acceptable; avoid repeated freeze–thaw.
• Aliquoting: Prepare single-use aliquots (10–50 µL) in low-bind tubes. Label with concentration and date.
• Thawing: Thaw on ice, mix gently by inversion or slow pipetting. Avoid vortexing to prevent shearing.
• Reconstitution (if lyophilized): Add nuclease-free water or TE to desired concentration; incubate 10–15 min on ice, flick gently to dissolve.
• Sterility: Use sterile, DNase/RNase-free tips and tubes. If required, 0.22 µm filter briefly; avoid filters that bind DNA.
• Documentation: Record lot, aliquot IDs, number of freeze–thaws, and working concentrations. Note: Concentration, volume, and buffer composition for this specific SKU are not specified; refer to CoA/Spec Sheet.

Structure and Identity

Item-specific facts (from Product Data)

• Product type: Lentiviral sgRNA construct targeting B2M (plasmid DNA reagent; exact map not provided)
• 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, SMILES, InChIKey, CID: Not applicable/not specified for plasmid DNA; refer to CoA/Spec Sheet.

General description (literature/typical for pLenti sgRNA vectors)

• Backbone: Lentiviral transfer plasmid enabling packaging into VSV-G–pseudotyped particles when co-transfected with packaging plasmids (e.g., psPAX2 + pMD2.G or equivalents).
• Expression cassettes: Typically includes a U6 or H1 promoter driving sgRNA (20-nt guide + tracrRNA scaffold). Many backbones also include an antibiotic selection marker (e.g., puromycin) and a fluorescent reporter; actual features for this item are not specified.
• Target: sgRNA designed to direct Cas9 to the human B2M (beta-2-microglobulin) locus for gene disruption or modulation; actual guide sequence not specified for this item.
• 2D “structure” in words: Circular double-stranded DNA plasmid containing a lentiviral LTR–Ψ packaging signal region, promoter–sgRNA scaffold unit, selectable marker cassette, and bacterial origin of replication/selection marker for propagation in E. coli (exact arrangement not specified).

Synthetic Utility

In the context of molecular biology, “synthetic utility” refers to how the construct enables genetic manipulations (literature/typical):

• Genome editing: Provides an sgRNA expression module that programs Cas9 nuclease to introduce double-strand breaks at B2M, enabling NHEJ-mediated indels and functional knockouts.
• Modular cloning: sgRNA cassettes are often interchangeable via Type IIS sites (e.g., BsmBI/BbsI), allowing rapid retargeting. Ensure the backbone’s cloning sites and overhangs are confirmed for this item (not specified here).
• Delivery flexibility: Supports transient plasmid transfection or stable integration via lentiviral transduction for long-term expression in dividing and non-dividing cells.
• Combinatorial studies: Can be paired with donor templates for HDR at B2M or multiplexed with additional sgRNAs using dual U6/H1 cassettes (if backbone supports it; not specified for this item).
• Readouts: Compatible with flow cytometry (MHC I staining), targeted amplicon sequencing for indel profiling, and functional assays relying on antigen presentation. Note: Backbone map, selectable marker, and cloning sites for this specific SKU are not provided; verify with CoA/Spec Sheet before planning experiments.

Target Specificity

Item-specific targeting details

• sgRNA target sequence, PAM context, species reactivity, and off-target assessment: Not specified for this item; refer to CoA/Spec Sheet.

General information (literature)

• Intended gene: Human B2M (beta-2-microglobulin). Effective guides typically target early exons to maximize frameshift probability, adjacent to a Cas9 PAM (e.g., NGG for SpCas9).
• Specificity considerations: Use in silico tools (e.g., CRISPOR, Benchling) to evaluate potential off-targets; verify edits by targeted sequencing. For cross-species use, confirm guide matching to the orthologous sequence.
• Controls: Include non-targeting sgRNA and a guide against a known essential gene for assay validation.