pLenti-MACROH2A2-sgRNA

Cat. No.: P746608

Specifications

Product Name

pLenti-MACROH2A2-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, recommended dilutions/amounts, and positive controls are not specified for this item; refer to CoA/Spec Sheet.

Note: General protocols for transfection, lentiviral packaging, and CRISPR validation are described under Reaction Conditions for contextual guidance only and are not item-specific instructions.

Biological Roles

This section provides general biological context for the named target MACROH2A2 and CRISPR applications; it is not an item-specific performance claim.

• Target gene (general): MACROH2A2 encodes a variant histone H2A with a C-terminal macrodomain implicated in chromatin organization, transcriptional regulation, and cellular differentiation. MacroH2A variants are associated with facultative heterochromatin and X-chromosome inactivation dynamics (literature).
• Functional highlights (literature/general):
  • Macrodomains can bind ADP-ribose moieties, linking macroH2A variants to ADP-ribosylation signaling pathways.
  • Incorporation of MACROH2A2 into nucleosomes influences nucleosome stability and can modulate accessibility to transcription factors and remodeling complexes.
• Research applications (general):
  • CRISPR-mediated disruption of MACROH2A2 facilitates studies on epigenetic regulation, lineage commitment, senescence-associated chromatin changes, and responses to DNA damage.
  • Comparative analyses of MACROH2A1 vs MACROH2A2 aid in dissecting isoform-specific roles in chromatin states.

Notes:

• Species specificity, sgRNA sequence, and on/off-target profiles for this item are not specified; consult the CoA/Spec Sheet and perform independent validation in the relevant model system.
• All uses are for research only; no clinical or diagnostic applications are implied.

Buffer Applications

As a plasmid DNA reagent, classical buffer “applications” (pH buffering of reactions) are not the primary focus. However, buffer choice is critical for DNA stability and downstream enzymology.

• Item-specific buffer: Not specified for this item; refer to CoA/Spec Sheet.
• General guidance (literature/practice):
  • Long-term storage: TE buffer (e.g., 10 mM Tris-HCl, 0.1–1 mM EDTA, pH ~8.0) can improve stability by chelating Mg2+ and inhibiting nucleases.
  • Enzymatic reactions (e.g., restriction digest, ligation, PCR): Use manufacturer-supplied reaction buffers; avoid EDTA carryover where it chelates required cofactors.
  • Cell transfection: Many protocols favor nuclease-free water or low-ionic buffers immediately before complexation with transfection reagents to optimize complex formation.
  • Electrophoresis: Standard TAE or TBE for agarose gels; use safer stains in place of ethidium bromide when possible.

Practical tips (general):

• Filter-sterilize solutions (0.22 µm) where sterility is needed; always use nuclease-free consumables.
• Avoid repeated freeze–thaw; prepare single-use aliquots in the chosen buffer.

Exact buffer formulations and concentrations for this item are not provided here.

Green Alternatives

While plasmid DNA products are not chemical solvents, greener practices can still be applied to their use and associated workflows.

• Considerations (general):
  • Reduce solvent use in plasmid preparation by selecting mini/midi prep kits with lower isopropanol/ethanol volumes or adopting membrane-based, water-wash protocols where validated.
  • Replace hazardous ethidium bromide with safer DNA stains (e.g., SYBR Safe) for gel analysis.
  • Prefer non-viral delivery (e.g., electroporation, RNP transfection) when biosafety, waste, or scalability make viral packaging less sustainable, balancing efficiency and cell-type constraints.
• Consumables and waste:
  • Use refillable filter tips and recyclable plastics where permitted; segregate GMO waste properly to avoid over-treatment of general waste streams.
  • Decontaminate with DNA-safe reagents to minimize chlorine load where acceptable and effective.

Comparison snapshot (general):

• Viral packaging vs RNP delivery:
  • Viral packaging enables stable integration and broad tropism but generates BSL-2 waste and uses transfection reagents and culture plastics.
  • Cas9 RNPs or non-viral plasmid transfection avoid viral waste streams and may reduce off-target dwell time, but often have shorter expression windows and may require optimization per cell type.

Item-specific greener alternatives are not defined; tailor choices to your facility’s EHS guidance.

Pharmaceutical Uses

This product is supplied strictly for research use only (per Product Data). No clinical, diagnostic, or therapeutic uses are claimed or supported.

• Excipient/formulation roles: Not applicable.
• Regulatory/pharmacopeial status: Not applicable for research-grade plasmid DNA constructs.

Context (general):

• Plasmid and lentiviral vectors can be components of investigational platforms in preclinical research, but transition to clinical-grade materials requires GMP manufacturing, comprehensive characterization (sequence-confirmed master cell banks, endotoxin/bioburden limits), and regulatory filings—none of which are implied for this research-use product.

For any application beyond basic research, consult your regulatory team; this product is not intended for human or veterinary use.

Physical Properties

For DNA plasmid reagents, conventional small-molecule physical constants (bp/mp, density, refractive index) are not applicable.

• Item-specific properties
  • Concentration, buffer composition, A260/A280: Not specified for this item; refer to CoA/Spec Sheet.
  • Form/appearance: Not specified for this item; refer to CoA/Spec Sheet.
• General/literature properties for plasmid DNA solutions (for context; not item-specific):
  • State: Aqueous solution or lyophilized solid; nucleic-acid polymers do not have defined bp/mp.
  • Solubility: Freely soluble in nuclease-free water and TE buffer; sensitive to nucleases and repeated freeze–thaw.
  • UV absorbance: Peak at 260 nm (A260/A280 ~1.8–2.0 for pure DNA, literature).
  • pH sensitivity: Stable near neutral pH; depurination increases under acidic conditions (literature).
  • Stability: Enhanced by storage at −20 to −80°C in low-ionic-strength, EDTA-containing buffers (literature best practice).

Practical notes (general):

• Thaw on ice and keep cold to limit nuclease activity.
• Avoid vortexing; mix gently to prevent shearing of high–molecular-weight DNA.
• Use low-binding, nuclease-free plastics and sterile technique.

All numerical or item-specific values are not provided here and should be obtained from the item’s CoA/Spec Sheet.

Quality and Grades

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

Context for plasmid reagents (general):

• Plasmid preparations are commonly offered as research-grade, endotoxin-reduced, or transfection-grade DNA. These designations relate to residual host contaminants (endotoxin, proteins, RNA) and are important for sensitive mammalian cell transfection and viral packaging.
• Analytical characterization (general examples; not item-specific):
  • Purity assessment by A260/A280, agarose gel electrophoresis (supercoiled vs nicked species), and, when relevant, endotoxin quantification (EU/µg DNA).
  • Identity confirmation by restriction digest mapping and/or full plasmid sequencing.
• Stabilizers/buffers: DNA is often supplied in nuclease-free water or low-salt buffers (e.g., 10 mM Tris, 0.1–1 mM EDTA, pH ~8.0). Exact buffer for this item is not specified.

Implications for use (general):

• For high-efficiency lentiviral packaging or primary cell transfection, select transfection-grade, low-endotoxin DNA and verify supercoiled percentage.
• If downstream NGS or cloning is planned, confirm insert integrity and absence of recombination via sequencing before critical experiments.

Reaction and Applications

Applications below are general for pLenti sgRNA plasmids and provided for context; they are not item-specific performance claims.

• CRISPR/Cas9 genome editing (general):
  • Expression of an sgRNA targeting the MACROH2A2 locus to direct Cas9-mediated DNA cleavage and facilitate knockout, indel formation, or HDR when donor templates are provided.
  • Suitable for cloning of alternative protospacers via golden gate or ligation-based methods (vector-dependent; consult map).
• Lentiviral particle production (general):
  • Co-transfect packaging cells (e.g., HEK293T) with the sgRNA pLenti plasmid plus helper plasmids (e.g., gag/pol and VSV-G) to generate pseudotyped lentivirus for stable delivery to dividing and non-dividing cells (institutional BSL-2/2+ practices apply).
• Pooled or arrayed screening (general):
  • Incorporation into pooled CRISPR libraries for loss-of-function screening, with selection markers and barcodes when present (not specified for this item).
• Validation workflows (general):
  • Confirm on-target editing by T7E1, ICE/TIDE analysis, amplicon NGS, or Sanger sequencing; assess off-targets via GUIDE-seq or predicted site sequencing.

Practical tips (general):

• Use highly pure, endotoxin-low plasmid DNA for transfection/packaging.
• Verify sgRNA sequence and cloning junctions by sequencing prior to critical experiments.
• Optimize MOI for lentiviral transduction to achieve desired multiplicity (e.g., MOI ~0.3 for single-copy integration in screens).

Reaction Conditions

Conventional chemical reaction conditions are not applicable. The following are general biological workflow conditions for pLenti sgRNA plasmids; they are not item-specific recommendations.

• Transfection (general):
  • Cells: HEK293T for packaging; many lines for direct plasmid transfection.
  • Reagents: Lipid-based (e.g., PEI, lipofection) or electroporation; optimize DNA:reagent ratios per supplier recommendations.
  • DNA quality: Endotoxin-low, A260/A280 ~1.8–2.0; supercoiled fraction high (literature best practice).
• Lentiviral packaging (general):
  • Co-transfect sgRNA plasmid with packaging plasmids (gag/pol, rev) and envelope (e.g., VSV-G).
  • Collect supernatants ~48–72 h post-transfection; filter (0.45 µm). Concentrate if needed (e.g., PEG or ultracentrifugation) following BSL-2 procedures.
• Transduction (general):
  • Target MOI adjusted to application (screens often MOI ~0.3; single-gene editing may use higher MOI). Polybrene may enhance efficiency in some lines.
  • Selection: Apply appropriate antibiotic or marker-based selection if present in the vector (not specified for this item).
• Editing validation (general):
  • Assess indel rates via TIDE/ICE or amplicon NGS 48–96 h post-Cas9 expression; isolate clones if clonal lines are required.

Exact conditions for this item (promoters, markers, sequences, and optimal transfection parameters) are not specified; validate empirically.

Safety and Handling

• GHS/SDS information
  • Signal word: Not specified for this item; refer to SDS.
  • H-statements: Not specified for this item; refer to SDS.
  • GHS classification/pictograms: Not specified for this item; refer to SDS.
• General safety guidance for plasmid DNA reagents (not item-specific):
  • While purified DNA is typically not classified as hazardous, pLenti backbones are used to generate lentiviral particles; handle constructs under institutional biosafety policies (often BSL-2 for lentiviral work). The plasmid itself is not replication-competent without packaging systems.
  • Avoid aerosol generation; wear standard PPE: lab coat, gloves, and eye protection.
  • Use nuclease-free technique to prevent degradation and potential cross-contamination.
• Incompatibilities/handling cautions (general):
  • Avoid strong acids/bases and oxidants that can degrade nucleic acids.
  • Do not mix with non-sterile reagents; maintain aseptic technique for cell work.
• First aid (general, non-authoritative):
  • Skin/eye contact with buffer: Rinse with water. If irritation persists, seek medical attention.
  • Ingestion/inhalation: Seek medical attention as per institutional guidance.
• Waste disposal (general):
  • Dispose of DNA-containing materials according to local biosafety and institutional GMO rules; decontaminate with 10% bleach followed by water, or use approved DNA decontamination agents.

Always consult the product’s SDS and your institutional biosafety committee (IBC) for definitive risk assessment and procedures.

Solvent Selection

This product is a plasmid DNA reagent; conventional solvent selection for organic solutes is not applicable.

• Item-specific solvent/buffer: Not specified for this item; refer to CoA/Spec Sheet.
• General guidance (literature/practice):
  • Preferred media for dissolution/storage: nuclease-free water or TE buffer (10 mM Tris, 0.1–1 mM EDTA, pH ~8). EDTA chelates divalent cations to inhibit nucleases.
  • Working dilutions can be made in nuclease-free water or cell-culture–compatible buffers immediately prior to transfection or cloning.
  • Avoid high concentrations of divalent cations (Mg2+, Ca2+) and avoid repeated freeze–thaw to maintain integrity.

Comparison (general):

• Water vs TE: Water maximizes compatibility for enzymatic reactions sensitive to EDTA; TE enhances long-term stability. Choose based on immediate application.

Note: Exact buffer composition and concentration for this item are not specified; consult the CoA/Spec Sheet supplied with the product.

Storage and Reconstitution

• Item-specific storage and shipping (from Product Data):
  • Storage: Store at −80°C.
  • Shipped in: Dry ice packs + Cold packs.
• Reconstitution and handling:
  • Reconstitution buffer, concentration, and volume: Not specified for this item; refer to CoA/Spec Sheet.
  • General guidance (not item-specific): If lyophilized, reconstitute in nuclease-free water or TE buffer to the desired concentration, mix gently (avoid vortexing), and allow complete dissolution on ice.
• Aliquoting (general):
  • Prepare single-use aliquots to avoid repeated freeze–thaw cycles that can shear DNA and reduce performance.
• Thawing (general):
  • Thaw on ice; briefly spin down condensate before opening. Keep on ice during setup.
• Short-term storage (general):
  • For frequent use, −20°C storage may be acceptable for working aliquots; retain a master stock at −80°C to preserve integrity.
• Avoidance of contamination:
  • Use nuclease-free, low-retention tubes and filtered tips; work in a clean area to prevent DNase/RNase contamination.

For exact buffer composition, concentration, and any stabilizers provided with this specific SKU (P746608), consult the CoA/Spec Sheet.

Structure and Identity

Brief description: pLenti-MACROH2A2-sgRNA is a recombinant DNA construct intended for CRISPR/Cas9 guide RNA expression from a lentiviral plasmid backbone targeting the MACROH2A2 gene (general description; not item-specific specs).

• Item-specific identifiers
  • 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, CID, InChIKey, SMILES: Not applicable for macromolecular plasmids; not assigned.
• Construct class (general):
  • Backbone type (literature/general): Lentiviral (pLenti) plasmid enabling packaging into VSV-G–pseudotyped lentiviral particles when co-transfected with helper plasmids.
  • Cargo (general): Single guide RNA (sgRNA) cassette under a small RNA polymerase III promoter (e.g., U6) guiding Cas9 to MACROH2A2 locus (target designation inferred from product name).
• Structural features (general):
  • Circular double-stranded DNA plasmid, typically 7–12 kb for many pLenti sgRNA vectors (size not specified for this item).
  • Common functional modules may include: 5′/3′ LTRs, ψ packaging signal, antibiotic resistance marker, bacterial origin of replication, and sgRNA scaffold (presence/arrangement not specified for this item; consult map/sequence).
• 2D description in words (general):
  • Closed circular dsDNA with discrete transcriptional cassettes arranged sequentially on the backbone; sgRNA protospacer sequence cloned into a defined site upstream of the gRNA scaffold.

Note: Exact plasmid map, sequence, restriction sites, promoters, selection markers, and insert details are not specified for this item; refer to CoA/Spec Sheet.

Synthetic Utility

In the context of molecular biology, “synthetic utility” pertains to genetic engineering workflows rather than organic synthesis.

• Functional modules (general):
  • sgRNA expression cassette drives transcription of a user-defined protospacer fused to a gRNA scaffold to guide Cas9 to MACROH2A2.
  • Lentiviral backbone allows stable genomic delivery when packaged into viral particles with helper plasmids.
• Typical transformations (general):
  • Cloning: Insertion/replacement of the 20-nt protospacer via type IIS restriction enzyme sites (e.g., BsmBI/BbsI) or Gibson/SLiCE, depending on vector design.
  • Barcoding/selection: Addition of selectable markers or reporters (if supported by backbone; not specified for this item) to enable selection and tracking.
  • Derivatization: Swapping promoters or scaffolds (e.g., extended tracrRNA variants) to modulate expression and activity.
• Retrosynthetic value (general):
  • A pLenti sgRNA vector can serve as a modular donor for assembling multiplex sgRNA arrays or for subcloning guides into alternative delivery backbones (AAV, non-viral).

Note: Exact restriction map, antibiotic resistance, and sequence-level features for this item are not specified; obtain the verified sequence/map from the CoA/Spec Sheet before planning cloning steps.

Target Specificity

Only item-specific details are allowed here.

• Target designation from product name: MACROH2A2 (gene) sgRNA.
• Species reactivity, sgRNA sequence, PAM context, off-target analysis, and any clone- or vector-specific attributes: Not specified for this item; refer to CoA/Spec Sheet.

No further target details are provided in the Product Data.

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-MACROH2A2-sgRNA); cross-reactivity in untested species may exist but is not guaranteed. Contact technical support for specific species inquiries.