pRYG DNA

Cat. No.: P419360

Storage

Store at 2-8°C

Shipped In

Wet ice

Vue d’ensemble

Description

This supercoiled plasmid is a pUC19 derivative in which the 206 bp SphI-NarI fragment of pUC19 was replaced with a 245 bp SphI-AccI fragment upstream of the human adult beta-globin gene promoter. The 245 bp fragment contains a 54 bp alternating repeat of purine/pyrimidine sequence that constitutes a strong eukaryotic topoisomerase II cleavage site.  Cleavages within the repeat are readily detected in the absence of topoisomerase II inhibitors such as m-AMSA or VM-26.  The sequence of the plasmid is given in Spitzner et al (reference below).

Shipping&storage

The plasmid is shipped at ambient temperature and should be stored at 4°C.

Specifications

Conditions de stockage de stockage

Store at 2-8°C

Expédié en

Wet ice

Ce produit nécessite l'expédition en chaîne froide. Les services terrestres et autres services économiques ne sont pas disponibles.

Application Protocols

No item-specific, tested application protocols are provided in the Product Data. Refer to the CoA/Spec Sheet for any assay-specific guidance.

General example workflows (not item-specific)

• Mini-scale restriction check: Digest 300 ng plasmid with 2–3 diagnostic enzymes in 20 µL, 30 min at 37°C; analyze on 1% agarose gel in TAE.
• Colony screening: Touch a colony to a PCR mix (colony PCR) targeting junctions; 25–30 cycles with robust polymerase; confirm by Sanger sequencing.
• Transient transfection (mammalian, general): Use an appropriate transfection reagent, maintain DNA:reagent ratios per vendor instructions (e.g., 1 µg DNA per well of a 12-well plate); replace medium after 4–6 h if cytotoxicity observed. Specific promoter/host compatibility for pRYG is unknown.
• Plasmid preparation for sensitive assays: Perform endotoxin reduction and buffer exchange (spin column or magnetic bead cleanup) to nuclease-free water or low-EDTA buffer.

These are illustrative, literature-based procedures. Optimize based on your host system, vector features, and the supplied buffer/concentration on the CoA.

Biological Roles

This section addresses the role of plasmid DNA constructs in biological research. The exact sequence, gene content, and biological function of pRYG DNA are not specified for this item.

General/literature context (not item-specific)

• Plasmids are episomal, autonomously replicating dsDNA molecules used as expression or cloning vectors. In bacteria, they leverage host replication machinery via an origin of replication and can achieve copy numbers ranging from a few to hundreds per cell depending on the ori and regulation.
• In research systems, plasmids deliver genetic instructions for protein expression, gene silencing (e.g., shRNA cassettes), reporter readouts, or genome engineering tools (e.g., Cas9, base editors) when such cassettes are present.
• Selectable markers (e.g., antibiotic resistance) enable maintenance under selection; promoters and UTRs dictate expression strength and host specificity. None of these features are defined here for pRYG DNA.
• In mammalian cells, plasmids are typically non-integrating and transient unless specialized integration systems are employed. Expression duration and copy number depend on delivery method and regulatory elements.
• As biochemical macromolecules, plasmids follow central dogma processes when transcribed/translated in compatible hosts. Their roles are entirely determined by sequence composition and regulatory architecture.

For pRYG DNA, consult the item’s CoA/Spec Sheet for any disclosed functional modules to plan appropriate host systems and assays.

Buffer Applications

Buffering considerations are highly relevant for the stability and use of plasmid DNA. Specific buffer composition for pRYG DNA is not provided.

General/literature guidance (not item-specific)

• Storage buffers: TE (10 mM Tris-HCl, 0.1–1 mM EDTA, pH 8.0) provides nuclease protection via Mg2+ chelation. For enzyme-heavy workflows, nuclease-free water or low-EDTA Tris (≤0.1 mM EDTA) can improve compatibility.
• pH range: Tris buffers are effective in pH ~7.5–8.5 at room temperature; buffering capacity shifts with temperature (ΔpKa/ΔT ≈ −0.028 pH/°C for Tris; literature).
• Working dilutions: Prepare in nuclease-free water to minimize ionic strength; supplement with buffer only when needed for stability.
• Electrophoresis: Use 0.8–1.2% agarose in TAE or TBE. TAE provides faster migration and lower buffering capacity; TBE gives sharper bands and higher buffering capacity. Include supercoiled markers to interpret isoforms.
• Enzymatic reactions: Most restriction enzymes and polymerases are compatible with ≤10 mM Tris, ≤0.1 mM EDTA, and ≤50 mM Na/K salts; check manufacturer’s buffer recommendations.

Always verify the supplied buffer on the CoA/Spec Sheet for SKU P419360 to decide whether desalting or buffer exchange is needed prior to sensitive enzymatic steps.

Green Alternatives

Green chemistry comparisons in the conventional solvent sense do not directly apply to a plasmid DNA reagent. However, sustainability can be addressed at the workflow level.

General, non–item-specific guidance

• Buffer choices: Prefer aqueous, low-toxicity buffers (nuclease-free water or Tris/EDTA) and avoid phenol–chloroform unless necessary. Column- or magnetic bead–based cleanups reduce chlorinated solvent use.
• Cold-chain optimization: This item ships on wet ice; coordinate delivery to minimize re-icing events and reduce wastage. Where validated, short-term room-temperature shipment with stabilizers can reduce energy consumption, but is not specified for this item—follow the provided shipping/storage guidance.
• Plastic reduction: Use reusable cold blocks and switch to low-binding, recycled-content plastics when nuclease control allows. Pool reactions and scale appropriately to decrease consumables.
• Waste management: Neutralize bleach after DNA decontamination and segregate halogenated from non-halogenated waste when organic extractions are used.

For SKU-specific sustainability opportunities (e.g., buffer formulation, concentration to reduce shipping mass), consult the CoA/Spec Sheet or contact Technical Support.

Pharmaceutical Uses

No therapeutic or clinical claims are made for this product. It is designated for research use only.

General, non–item-specific context

• In pharmaceutical R&D labs, research-grade plasmid DNA can serve as a tool for assay development, screening, expression of research proteins, and analytical standards (e.g., reference templates for qPCR). Such uses are confined to preclinical research and process development.
• For any application in regulated manufacturing or clinical studies, specialized grades (e.g., GMP-compliant plasmid DNA with defined release specifications, impurity controls, and full traceability) are required. The grade for this item is not specified.
• Excipient/formulation roles: Plasmid DNA is not used as a conventional excipient. In advanced therapy development, plasmids may be intermediates for viral vector production or mRNA templates; this product is not qualified for such uses unless explicitly stated in its CoA/Spec Sheet.

Consult your quality unit and the CoA to determine suitability for any regulated workflow. Absent explicit qualification, restrict use to basic research.

Physical Properties

Item-specific (from Product Data)

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Concentration, buffer composition, A260/A280, endotoxin level: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight/formula: Not specified for this item; refer to CoA/Spec Sheet.

General/literature properties of plasmid DNA (non-spec for this item)

• Physical state: aqueous solution or lyophilized solid; highly water-soluble in low-salt buffers (e.g., TE). Viscosity increases with concentration and size (bp).
• UV absorbance: ε260 ≈ 0.020 (µg/mL)⁻¹·cm⁻¹; 1 A260 unit ≈ 50 µg/mL for dsDNA (literature). Typical purity ratios: A260/A280 ≈ 1.8–2.0; A260/A230 ≈ 2.0–2.4 (application-dependent) (literature). These values are general guidelines, not specifications for this item.
• pH stability: dsDNA is most stable near neutral pH (7–8.5). Acidic conditions promote depurination; strong base can denature DNA (literature).
• Thermal behavior: localized strand melting depends on GC content; prolonged exposure >65–70°C can nick or denature plasmids without protective salts (literature).
• Solubility: readily soluble in nuclease-free water and low-ionic-strength Tris buffers; precipitates in alcohol with monovalent cations (e.g., Na+, NH4+) (literature).

Always consult the item’s CoA/Spec Sheet for any measured properties of this lot; do not treat literature values as product specifications.

Quality and Grades

Item-specific (from Product Data)

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

How to interpret grade for plasmid DNA (general guidance; not item-specific)

• Common descriptors include: research-grade, transfection-grade (low endotoxin), sequencing-grade, and GMP-like or GMP-compliant (for manufacturing workflows). Each denotes specific upstream controls and downstream QC metrics.
• Typical QC attributes to review on the CoA: A260/A280 and A260/A230 ratios, concentration (ng/µL), isoform distribution (supercoiled % by agarose or capillary electrophoresis), residual RNA/protein, endotoxin (EU/µg), residual salts/solvents, host genomic DNA, and sterility/bioburden.
• Sequence identity and integrity: may be supported by restriction digest fingerprinting, Sanger or NGS coverage, and length (bp). For functional vectors, verification of antibiotic marker and key cassettes is standard.
• Stabilizers/buffers: TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) is commonly used to chelate Mg2+ and inhibit nucleases; nuclease-free water can maximize downstream compatibility but is less protective. Buffer for this item is not specified.

Always defer to the item’s CoA/Spec Sheet for definitive grade definitions and acceptance criteria applicable to SKU P419360.

Reaction and Applications

As a plasmid DNA preparation, pRYG DNA is intended for molecular biology workflows rather than small-molecule chemical reactions.

Item-specific (from Product Data)

• Manufacturer Applications: Not specified for this item; refer to CoA/Spec Sheet.

General/literature applications for plasmid DNA (not item-specific)

• Cloning and assembly: Restriction/ligation cloning, Gibson assembly, Golden Gate (Type IIS), SLIC, In-Fusion, and homologous recombination-based methods.
• Transformation/transfection: Introduction into competent E. coli for propagation; introduction into mammalian, yeast, or plant cells for research expression when appropriate promoters are present (features for this item are not specified).
• In vitro transcription/translation templates: Linearized plasmid as a template for T7/SP6 transcription systems (promoter presence not specified for this item).
• Control standards: qPCR/ddPCR standards, sequencing controls, or assay validation references when sequence is known.
• Protein or pathway engineering: As a backbone for inserting GOIs, tags, and regulatory parts; exact cassette content for pRYG is not provided.

Practical tips (general)

• Maintain supercoiled fraction: Minimize freeze–thaw and vortexing; use wide-bore tips for viscous, high-concentration stocks.
• For enzymatic reactions: Confirm buffer compatibility (salt/EDTA). If inhibition is suspected, perform cleanup (spin column, ethanol precipitation) before assembly or digest.

Reaction Conditions

Classical reagent “reaction conditions” are not directly applicable; instead, consider standard conditions for common enzymatic manipulations of plasmid DNA. The following are general literature guidelines and not specifications for this item.

• Restriction digestion: 0.2–1 µg plasmid DNA in 10–50 µL with 1× recommended buffer, 3–10 U enzyme per µg DNA, 37°C for 15–60 min (or enzyme-specific temperature). Avoid >10% glycerol to limit star activity. Heat-inactivate or purify before downstream use.
• Ligation (sticky/blunt): Molar insert:vector ratios 3:1 (sticky) or 5:1 (blunt); 1× ligase buffer with ATP; T4 DNA ligase at 16°C for 1–2 h or room temperature for 10–30 min; overnight at 4–16°C for difficult assemblies.
• Gibson/Isothermal assembly: 20–40 bp overlaps; incubate 50°C for 15–60 min. Use equimolar parts totaling 0.02–0.5 pmol DNA.
• PCR amplification from plasmid template: 10–100 ng template per 50 µL; annealing temperature per primer Tm; high-fidelity polymerase for cloning.
• Transformation into E. coli: Chemically competent cells (1–5 × 10^8 cfu/µg): 30 min on ice, 42°C heat shock 30–45 s, recover 1 h at 37°C in SOC. Electroporation: 1–2 mm cuvette, 1.8 kV, time constant ~4–5 ms; recover 1 h.
• Endotoxin-sensitive applications: Use low-endotoxin DNA and sterile technique; buffer exchange if necessary.

Adjust conditions based on enzyme supplier recommendations and the actual buffer of the supplied DNA (consult CoA/Spec Sheet).

Safety and Handling

Item-specific hazard data (from Product Data)

• 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 guidance (plasmid DNA reagents; for research use only)

• Expected hazards: Plasmid DNA in aqueous buffer is generally considered low hazard; however, follow institutional biosafety practices for recombinant DNA materials. Avoid inhalation of dried aerosols and contact with eyes/mucosa. Handle as BSL-1 unless the sequence confers additional risks; lacking sequence details, adopt conservative containment per your IBC.
• PPE: lab coat, gloves, and safety glasses. Change gloves frequently to prevent nuclease contamination.
• Incompatibilities: Nucleases (DNase) rapidly degrade DNA; use nuclease-free plasticware, tips, and buffers. Avoid microbial contamination.
• First aid (general): Rinse skin/eyes with water if contact occurs. If ingested or inhaled as aerosolized dust, seek medical attention according to institutional protocols. Always consult the SDS for authoritative instructions.
• Environmental: Do not release genetically modified organisms (GMOs) or recombinant DNA into drains. Decontaminate surfaces with 10% bleach followed by ethanol/water wipe.
• Transport: Shipped on wet ice; keep cold chain intact to minimize nuclease activity.

Refer to the SDS and your institutional biosafety committee (IBC) for risk assessment tailored to your application and organism/sequence context.

Solvent Selection

This product is a DNA reagent, not a small-molecule solute, so classical organic solvent selection is generally not applicable.

Item-specific (from Product Data)

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

General/literature guidance for plasmid DNA

• Preferred media: nuclease-free water or low-ionic-strength Tris buffers (e.g., 10 mM Tris-HCl, pH 7.5–8.5) with optional 0.1–1 mM EDTA to chelate divalent cations and suppress DNase activity.
• Avoid: strong acids/bases, oxidants, and most organic solvents (phenol, chloroform, acetone) unless used in controlled extraction workflows. Alcohols (ethanol/isopropanol) precipitate DNA in the presence of monovalent cations.
• Compatibility: Downstream enzymology (PCR, restriction digest, ligation, Gibson, assembly) typically tolerates ≤1–10 mM Tris and ≤0.1 mM EDTA in the reaction; high salt or EDTA can inhibit enzymes.

Comparison (general)

• TE buffer: maximizes stability during storage; may require dilution or cleanup before enzyme reactions.
• Nuclease-free water: maximizes downstream compatibility; slightly less stable for long-term storage compared to TE.

Consult the CoA to determine the exact buffer and adjust your workflow (e.g., dilution or cleanup) accordingly.

Storage and Reconstitution

Item-specific (from Product Data)

• Storage conditions: Store at 2–8°C.
• Shipped in: Wet ice.

General handling guidance for plasmid DNA (not item-specific)

• Short-term (days–weeks): 2–8°C is suitable, especially in TE buffer. Avoid repetitive freeze–thaw cycles.
• Medium/long-term: For multi-month storage, aliquot and freeze at −20°C or −80°C in TE to preserve supercoiling. Thaw gently on ice and mix by slow pipetting; avoid vortexing to limit nicking.
• Reconstitution: If received lyophilized (not specified for this item), dissolve in nuclease-free water or TE to desired concentration. Allow complete dissolution on ice for 10–15 min with gentle mixing.
• Working stocks: Prepare low-concentration working aliquots to minimize contamination and shear; store on ice during bench work.
• Contamination control: Use DNase/RNase-free consumables. If sterility is required, 0.22 µm filter using low-protein-binding membranes; avoid UV exposure that can damage DNA.
• Documentation: Record lot number, concentration (A260-based), buffer composition, and number of freeze–thaw cycles.

For any deviations from these general practices or to confirm supplied buffer/concentration, consult the item’s CoA/Spec Sheet for SKU P419360.

Structure and Identity

Item-specific (from Product Data)

• Product name: pRYG DNA (SKU: P419360)
• 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/Not specified for this item; refer to CoA/Spec Sheet.

General/literature description of form and features (plasmid DNA)

• pRYG DNA is expected to be a circular, double-stranded DNA (dsDNA) construct (plasmid), typically propagated in E. coli. As a biopolymer, it does not have a single exact molecular formula or a conventional SMILES/InChI representation used for small molecules.
• 2D/primary structure: A covalently closed circular dsDNA comprising a bacterial origin of replication, one or more selection markers, and an application cassette (e.g., promoter–gene–terminator). Exact sequence, size (bp), antibiotic marker(s), and cloning sites for this item are not specified.
• Functional domains (general): origin of replication (e.g., ColE1-type), selectable marker (e.g., antibiotic resistance), multiple cloning site (MCS), and optional regulatory elements (promoters, terminators, tags). Presence/identity of any of these in pRYG is not specified.

Structural considerations (general)

• Topology: supercoiled, nicked circular, and linear isoforms may be present depending on preparation; supercoiled is typically dominant in high-quality preparations.
• Sequence identity: For sequence-level identity and restriction map, consult the item’s CoA/Spec Sheet. Without sequence disclosure, pRYG should be treated as an unspecified plasmid backbone for general cloning or expression tasks.

Synthetic Utility

This section reframes “synthetic utility” for genetic engineering workflows rather than small-molecule synthesis.

Item-specific

• Sequence features, restriction map, and cloning sites for pRYG DNA: Not specified for this item; refer to CoA/Spec Sheet.

General/literature utility of plasmid backbones (not item-specific)

• Vector scaffold: Provides origin of replication and selectable marker to propagate engineered inserts. Acts as a modular chassis for promoters, coding sequences, tags, and regulatory parts.
• Assembly modalities: Compatible with enzyme-based cloning (restriction/ligation), homologous recombination-based methods (Gibson, In-Fusion, NEBuilder), and type IIS–based hierarchical assembly (Golden Gate/MoClo). Strategic design of overhangs enables scarless junctions.
• Functional diversification: The same backbone can host libraries (mutagenesis, directed evolution), multigene cassettes (operons), or regulatory circuits. Backbone size and copy number influence transformation efficiency and expression burden.
• Analytical leverage: Serves as a quantitative DNA standard (when sequence known) for qPCR/ddPCR calibration and NGS control spike-ins.

Before planning a build, consult the pRYG DNA map (if available) to choose restriction enzymes that avoid essential features and to define homology arm placements.

Target Specificity

Not applicable. This product is a plasmid DNA preparation, not an antibody or affinity reagent. No antigen, epitope, or species reactivity information is relevant.

• Item-specific data: Not specified for this item; refer to CoA/Spec Sheet if any sequence-target interactions are provided for assay controls.