DNA Detection Kit (Diphenylamine, Colorimetric Method)

Cat. No.: D1528847

GRADE & PURITY BioReagent

개요

This kit is based on the classical diphenylamine colorimetric method for the quantitative determination of deoxyribonucleic acid (DNA) in various samples. Under strong acidic and heating conditions, the α-deoxyribose in DNA undergoes dehydration and isomerization to form ω-hydroxy-γ-ketopentanal, which specifically reacts with diphenylamine reagent to generate a stable blue-colored complex. The chromogenic product exhibits a maximum absorbance at 595 nm. Within the DNA concentration range of 20–400 μg/mL, the absorbance shows a good linear correlation with DNA concentration, enabling accurate quantification of DNA content. The addition of trace amounts of acetaldehyde to the reaction system significantly enhances detection sensitivity, lowers the detection limit, and improves both accuracy and reproducibility. This kit offers advantages including simple operation, low cost, and good stability. It is suitable for the determination of total DNA content in animal and plant tissues, microorganisms, and cultured cells, and is widely used in biochemistry, molecular biology, and general research and teaching laboratories. For research use only. Not for clinical diagnosis, food testing, or other non-research applications.

Specifications

사양 및 순도

BioReagent

등급

BioReagent

보관 및 배송

보관 조건

Store at -20°C

배송

Ice chest + Ice pads

Application Protocols

Follow the kit’s official insert for validated steps. The outline below is provided as general, literature-based guidance for planning and training; adapt only within your lab’s method validation.

General diphenylamine DNA assay workflow (literature)

1. Preparation

• Equilibrate reagents to room temperature, then protect the DPA reagent from light. Preheat a water bath or dry block to ~95–100 °C.
• Prepare a DNA standard curve (e.g., 0–200 µg per reaction) using a suitable DNA standard in the same matrix as samples. Include a reagent blank.

2. Reaction setup

• Aliquot sample or standard into acid-resistant tubes or plate wells.
• Add the DPA reagent at a fixed sample:reagent volume ratio across all tubes (commonly large excess of reagent). Mix gently; avoid bubble formation.

3. Color development

• Cap/cover tightly. Heat at ~95–100 °C for 10–30 minutes (optimize for your setup). Ensure uniform heating; use tube floats/racks.
• Cool to room temperature (ice bath if needed) before opening to minimize aerosol/splash.

4. Measurement

• If turbidity is present, briefly centrifuge to clarify.
• Transfer to compatible cuvettes or read directly in plates. Measure absorbance near 595–600 nm versus the reagent blank.

5. Quantification and QC

• Plot A versus DNA mass/concentration; use the linear region for quantitation.
• Assess recovery with a matrix spike; acceptable recovery ranges depend on your internal criteria.

Notes

• Item-specific volumes, λmax, and timing are not specified here; refer to the kit insert. Handle all acidic reagents with appropriate PPE in a fume hood.

Biological Roles

Scope note: This section provides general biochemical context for the analyte (DNA) detected by the kit; it does not describe any medical or diagnostic use.

General/literature background

• DNA is the primary carrier of hereditary information in cells, composed of deoxyribonucleotides linked via phosphodiester bonds forming polynucleotide chains with 2′-deoxyribose sugars.
• The assay targets the deoxyribose moiety after acid-catalyzed depurination/dehydration, enabling relative specificity for DNA over RNA.
• Cellular distribution: DNA is primarily nuclear (chromosomal) and mitochondrial in eukaryotes; prokaryotes house genomic DNA in nucleoids with plasmids as extrachromosomal elements.
• Turnover and processing: DNA replication, repair, and recombination pathways dynamically remodel DNA. Chemical detection of DNA content can serve as a proxy for cell number or biomass in research contexts.
• Matrix considerations: Plant tissues contain polyphenols and polysaccharides that can co-extract with DNA and potentially interfere with colorimetric detection unless removed; protein-dense animal tissues may require robust deproteinization.

Practical implications for this assay

• Because signal arises from deoxyribose chemistry, extensively degraded DNA can still contribute to signal provided deoxyribose is present; however, depurination state and fragment size can influence kinetics.
• Nucleotides and dNMPs/dNTPs may yield lower responses than high-molecular-weight DNA on a mass basis (literature), emphasizing the importance of matrix-matched standards when quantifying partially hydrolyzed samples.

Buffer Applications

This product is not a buffer system; it is a colorimetric DNA detection kit. Nevertheless, buffer considerations can influence assay performance.

Item-specific buffer composition: Not specified for this item; refer to kit insert.

General/literature guidance

• Pre-assay sample buffers: Low ionic strength aqueous buffers (e.g., 1–10 mM Tris, pH ~7–8) with EDTA can stabilize DNA prior to assay. Avoid high concentrations of primary amines (e.g., Tris >100 mM) as they may react with aldehydic intermediates under strong acid.
• Salt effects: High salt (e.g., >0.5 M NaCl) generally has minor direct effects after strong acid addition but can influence precipitation during heating; clarify samples if turbidity arises.
• Detergents/chaotropes: Carryover of SDS, CTAB, guanidinium, or phenol from extraction protocols can elevate blanks. Where possible, precipitate/clean DNA before color development or use matrix-matched standards.
• Deproteinization: For tissue lysates, pre-clear with perchloric or trichloroacetic acid (literature) and neutralize/decant prior to adding DPA reagent to reduce protein-derived background.

Recommendation

• Validate your routine sample buffer for compatibility by preparing a DNA standard curve in that exact buffer and comparing slope/blank to a water-based curve.
• If precipitation occurs upon adding the DPA reagent, reduce sample volume fraction or dilute the sample buffer before the reaction step.

Green Alternatives

Perspective on greener choices focuses on safety, waste, and resource use, balanced against analytical performance.

General comparison (literature; not item-specific)

| Approach | Hazard profile | Sensitivity | Selectivity for dsDNA | Instrumentation | Notes | |---|---|---|---|---|---| | Diphenylamine colorimetry (this kit’s principle) | Uses strong acids and aromatic amine; corrosive and irritant risks; modest organic content | Moderate | Good vs RNA | Basic spectrophotometer (VIS) | Robust, inexpensive; heat required; generates acidic organic waste | | UV A260 | Minimal reagents; greenest in terms of chemical use | Low–moderate | Poor (protein/phenol interfere) | UV spectrophotometer | Fast, no hazardous waste; needs purified samples | | Fluorescent dyes (e.g., PicoGreen, Hoechst) | Dyes often hazardous to aquatic life; light-sensitive; typically low volumes | High | High (dsDNA-specific for some dyes) | Fluorometer/plate reader | Lower waste per assay but requires specialized instruments | | Diphenylamine substitutes (e.g., indole-based reagents, literature) | Varies; some reduce strong-acid use | Moderate | Variable | VIS | Less standardized, may still need acid/oxidant |

Greening tips for DPA workflows (general)

• Microscale: Run assays in 96-well plates with minimal volumes to reduce acidic waste.
• Containment: Use sealed tubes during heating to minimize emissions; cool before opening.
• Waste: Neutralize acids, segregate amine-containing organics, and use institutional hazardous waste streams.
• Energy: Prefer precise dry blocks over prolonged boiling baths to reduce energy/time.

When greener priority dominates and sensitivity allows, UV A260 on well-purified DNA is the lowest-chemical-footprint method. For complex matrices requiring specificity, fluorescent dyes can reduce acid use at the cost of dye hazards and instrument needs.

Pharmaceutical Uses

This product is for research use only and is not intended for clinical or diagnostic applications.

General/literature context (non-clinical)

• Process development and in-lab QC: Colorimetric DNA assays have been used in research settings to estimate residual host-cell DNA or total DNA content in bioprocess intermediates, filter retentates, or laboratory-scale purification workflows. Such use requires internal qualification and should be complemented by orthogonal methods for regulated work.
• Raw material assessment: Researchers may use DPA-based assays to screen crude extracts (e.g., plasmid preps, lysates) for DNA content before proceeding to downstream steps such as enzymatic treatments or chromatography.
• Excipient/formulation role: None. This kit is an analytical reagent set and does not serve as an excipient or formulation aid.

Compliance and documentation

• No pharmacopeial status is claimed for this product. If pharmacopeial or regulatory compliance is required, adopt compendial methods (where applicable) and validate performance characteristics (specificity, linearity, accuracy, precision, robustness) in your laboratory.

Note

• For any use in manufacturing support or QC, consult your QA unit and perform method validation suitable to the stage of development. This kit provides an enabling chemistry but not a validated regulatory method per se.

Physical Properties

Because this is a multi-reagent kit, item-specific physical constants are not provided here.

Item-specific specs

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Density, refractive index, pKa, logP, solubility: Not specified for this item; refer to CoA/Spec Sheet.
• UV/Vis characteristics of the working assay solution: Not specified for this item; refer to CoA/Spec Sheet.

General/literature information relevant to the assay chemistry (not item-specific)

• Chromophore absorbance: The Dische diphenylamine reaction typically yields a blue product with a maximum near 595–600 nm (literature; some formulations report 590–620 nm depending on acid/oxidant).
• Solubility context: DNA is measured in aqueous samples; the reaction medium is strongly acidic (commonly concentrated acetic and/or sulfuric acid in literature), in which the DPA reagent is soluble and DNA undergoes depurination/dehydration.
• Temperature: Color development is typically accelerated by heating (e.g., near boiling water bath, ~90–100 °C in literature). The exact temperature/time for this product is not specified here.

Practical notes

• Use matched optical plastics or glass compatible with strong acids and visible absorbance near 600 nm. Avoid polystyrene cuvettes if concentrated acids are present. Rinseware thoroughly to prevent background color.
• For quantitative work, establish a calibration curve with DNA standards prepared in the same matrix as samples.

Quality and Grades

• Item-specific grade: BioReagent. This designation typically indicates components and packaging suitable for general biological/biochemical research workflows. It does not imply clinical/diagnostic suitability.
• What BioReagent grade implies (general):
  • Sourcing and handling geared for lab research reliability.
  • Low background in routine colorimetric/biochemical assays compared with technical grades.
  • Batch-to-batch consistency appropriate for quantitative work, with QC tests relevant to the intended application.
• What is not implied: Absence of every possible trace impurity, GMP compliance, or suitability for in vitro diagnostics. For regulated applications, request additional documentation and perform in-house qualification.
• Stabilizers/antioxidants: Not specified for this item; refer to CoA/Spec Sheet.
• DNA standard or controls: Not specified for this item; refer to CoA/Spec Sheet.

Practical guidance for users

• Verify performance with an in-house DNA standard curve (same matrix as samples) upon opening a new lot.
• Record lot numbers and retain CoA. If your workflow relies on absorbance at a specified wavelength, confirm the kit’s recommended λmax and bandwidth with your spectrophotometer and validate linear range.
• If using the kit in high-throughput settings, prequalify plastics and pipette tips for acid compatibility and low background.

For detailed acceptance criteria (e.g., blank absorbance tolerance, linearity range, inter-assay CV), consult the kit insert or request a specification sheet.

Reaction and Applications

This kit implements the Dische diphenylamine reaction for DNA quantification.

General/literature mechanism (not item-specific)

• Under strong acid, DNA depurinates, and the deoxyribose moiety dehydrates to form an aldehydic intermediate (e.g., furfural-like species). Diphenylamine condenses with this intermediate, and in the presence of an oxidant forms a conjugated cationic chromogen absorbing near 590–600 nm. RNA (ribose) reacts far less intensely, providing selectivity for DNA.

Typical applications (research use only)

• Quantitative determination of DNA in cell/tissue lysates, isolated nuclei, or plant materials where UV A260 is confounded by protein or phenolics.
• Monitoring DNA during extraction, hydrolysis, or nuclease treatments.
• Estimating total DNA content in bioprocess samples (e.g., downstream impurity monitoring) as part of in-lab research QC.

Practical tips (general)

• Calibration: Prepare a standard curve with calf thymus DNA or a matrix-matched DNA standard covering your expected range; include a reagent blank.
• Sample prep: Deproteinize turbid or protein-rich matrices to reduce background; avoid carryover of phenol/guanidinium from extraction kits.
• Heating: Maintain consistent development temperature/time (e.g., controlled water bath or dry block). Small variations can lead to significant absorbance differences.
• Wavelength: Verify the λmax for your specific formulation. Read at the recommended wavelength using matched cuvettes or plate wells.
• Interferences: Monosaccharides, strong nucleophiles (e.g., hydroxylamine), and some aldehyde-containing contaminants can contribute to background; keep these low.

Item-specific directions for volumes, times, and λmax are not provided here—follow the kit insert for validated conditions.

Reaction Conditions

Item-specific validated conditions are not provided here; consult the kit insert for definitive instructions. The following summarizes common literature conditions for the Dische diphenylamine DNA assay to aid planning and understanding.

General/literature conditions

• Reagent composition: Diphenylamine dissolved in concentrated acetic acid with added sulfuric acid and an oxidant (e.g., ferric chloride or acetaldehyde as enhancer). Exact ratios vary by protocol.
• Temperature and time: Heat reactions at ~90–100 °C for 10–30 minutes (water bath or dry block). Some protocols recommend longer times at lower temperatures for convenience; kinetics are temperature-dependent.
• Wavelength: Measure absorbance near 595–600 nm against a reagent blank. Confirm λmax for your formulation by scanning 500–700 nm on a DNA standard.
• Stoichiometry: Excess DPA relative to deoxyribose to drive chromophore formation; maintain consistent sample-to-reagent volume ratios across standards and unknowns.
• Linear range: Typically low microgram to low milligram DNA per reaction (literature), depending on pathlength and formulation. Establish the linear dynamic range empirically with your setup.
• Interferences: RNA contributes weakly; sugars and aldehyde-generating contaminants can elevate background. Protein and particulates cause turbidity—clarify by centrifugation if needed after color development and before reading.

Practical controls

• Include a reagent blank, at least five DNA standards spanning your expected range, and a matrix spike recovery to gauge interference.
• Verify day-to-day reproducibility by running a mid-range control sample across assays.

Safety and Handling

Always consult the product SDS and kit insert for authoritative safety guidance. The following is general, literature-based information for diphenylamine DNA colorimetric assays and is not a substitute for the SDS.

Item-specific hazard data

• GHS classification, pictograms, signal word, H-statements: Not specified for this item; refer to SDS/kit insert.
• Storage conditions (item-specific): Store at −20 °C.
• Shipping (item-specific): Ice chest + ice pads.

General/literature safety considerations for DPA DNA assays

• Corrosivity: Traditional DPA formulations employ strong acids (e.g., sulfuric and/or acetic acid). These are corrosive; use a chemical fume hood, acid-resistant gloves, splash goggles/face shield, and lab coat.
• Toxicity: Diphenylamine is harmful if swallowed or inhaled and can be irritating to skin/eyes; avoid aerosol and dust formation. Many DPA reagents include oxidants (e.g., ferric salts) that may enhance irritation/oxidation hazards.
• Incompatibilities: Avoid contact with bases, strong oxidizers/reducers outside intended formulation, and reactive metals when strong acids are present. Do not mix with bleach or nitrosating agents.
• Heat: Color development often requires heating (near 100 °C); use heat-resistant racks, secondary containment, and acid-compatible sealed tubes to mitigate pressure buildup and splashing.
• Waste: Neutralize and segregate acidic, aromatic amine–containing waste per institutional protocols. Label waste with “acidic organic/amine-containing”.

First-aid overview (general)

• Skin/eye contact with acids/DPA: Immediate water rinse for ≥15 minutes; remove contaminated clothing; seek medical attention.
• Inhalation: Move to fresh air; support breathing as needed; obtain medical attention.
• Ingestion: Rinse mouth; do not induce vomiting; seek medical attention.

Defer to the SDS for specific PPE, exposure limits, and spill response.

Solvent Selection

As an assay kit, solvent selection refers to sample matrix compatibility and the reagent medium rather than choosing a standalone solvent.

Item-specific details

• Composition of the working reagent(s): Not specified for this item; refer to kit insert.

General/literature guidance for the DPA colorimetric assay

• Reaction medium: Strongly acidic, typically a mix of concentrated acetic and/or sulfuric acid with diphenylamine and an oxidant (literature). This medium dehydrates deoxyribose to reactive aldehydes that condense with DPA.
• Sample matrices:
  • Aqueous buffers (e.g., TE, Tris, phosphate) are generally tolerated but high concentrations of primary amines or strong nucleophiles can interfere by reacting with aldehydes.
  • Detergents and phenolic extractants may increase background. Protein-rich samples may require deproteinization (e.g., perchloric or trichloroacetic acid precipitation, literature) prior to assay.
  • Organic co-solvents should be minimized; ethanol/isopropanol residues from nucleic-acid precipitation can affect color development if present in high amounts.
• Plastics and glassware:
  • Use acid-resistant plastics (PP, PTFE) or borosilicate glass; avoid polystyrene when strong acids are present.
• When to choose this kit vs alternatives:
  • Choose DPA colorimetry for robust endpoint quantification of DNA when a UV spectrophotometer and heating block/water bath are available and fluorescent readers are not desired.
  • Consider fluorescent dyes (e.g., PicoGreen, Hoechst; literature) for higher sensitivity or complex matrices, acknowledging instrumentation and safety tradeoffs.

Comparison snapshot (general)

• DPA colorimetry: Visible readout (~600 nm), tolerant of modest turbidity after blanking, moderate sensitivity, heat/acid required.
• A260 UV: Rapid, non-destructive, but poor specificity in the presence of protein/phenol.
• Fluorescent dyes: Highest sensitivity/specificity but require fluorescence-capable instrumentation and dark handling.

Storage and Reconstitution

Item-specific guidance

• Storage temperature: Store at −20 °C.
• Shipping: Ice chest + ice pads.
• Appearance and reconstitution instructions: Not specified for this item; refer to CoA/Spec Sheet and kit insert.

General handling recommendations for DPA DNA assay kits (literature; not item-specific)

• Light sensitivity: Protect diphenylamine-containing reagents from light to minimize baseline drift due to slow oxidation.
• Freeze–thaw: If the kit provides concentrated or ready-to-use liquid reagents, aliquot upon first thaw to minimize freeze–thaw cycles that can alter background color or precipitate components. Mix thoroughly after thawing to re-dissolve any separates.
• Temperature equilibration: Warm refrigerated/frozen reagents to room temperature before use unless the protocol specifies otherwise; cold, viscous acid mixtures can pipette inaccurately.
• Container compatibility: Store acidic reagents in compatible, vented containers to avoid pressure buildup. Open slowly and away from face in a fume hood.
• Stability: For working solutions prepared from concentrates, note the on-bench stability window (often hours to a day for DPA reagents, literature) and discard if background absorbance rises or color appears.
• Record-keeping: Track lot numbers, preparation dates, and any dilutions; include control charting of blank/standard absorbance to monitor reagent health over time.

For definitive shelf-life and reconstitution information, follow the kit insert and CoA/Spec Sheet.

Structure and Identity

Brief overview: This is a research-use DNA detection kit based on the classic Dische diphenylamine (DPA) colorimetric reaction for deoxyribose. It is a multi-component kit, not a single defined molecule.

• SKU: D1528847
• Product name: DNA Detection Kit (Diphenylamine, Colorimetric Method)
• Item type: Multi-reagent assay kit (colorimetric)
• Grade/Purity (item-specific): BioReagent
• Molecular formula, Molecular weight, SMILES, InChIKey (item-specific): Not specified for this item; refer to CoA/Spec Sheet.
• Structural features (general/literature): The key chromogenic reagent is diphenylamine (DPA), an aniline-derived aromatic amine. Under strong acidic and dehydrating conditions, depurinated deoxyribose from DNA forms an aldehydic intermediate that condenses with DPA to give a blue quinonediimine-type chromophore.
• 2D structural description (general/literature): Diphenylamine consists of two phenyl rings bound to a central secondary amine (Ph–NH–Ph). The formed chromophore in the assay is an oxidized conjugated species absorbing in the visible range (~590–620 nm, literature), but the exact structure depends on reagent composition and oxidant.

Notes

• Component identities, exact concentrations, and stabilizers of this commercial kit are not disclosed here. Consult the kit insert and CoA for definite identities and any auxiliary reagents included (e.g., acid mixture, oxidant, standards).

Synthetic Utility

Not a building block or synthetic reagent. This product is an analytical kit for DNA quantification and does not directly participate in preparative organic synthesis.

General/literature note (for context only)

• The diphenylamine reaction exemplifies acid-catalyzed dehydration of deoxyribose followed by electrophilic aromatic substitution/condensation to form a conjugated chromophore. While mechanistically interesting to synthetic chemists, this transformation is not typically harvested for preparative synthesis due to complex product mixtures and the requirement for strong acid/oxidant conditions.

Guidance for users

• If your workflow involves chemical DNA modifications (e.g., depurination, sugar cleavage), be aware that such treatments may alter color yields in DPA assays. Validate the assay response versus your specific modified DNA or rely on orthogonal quantification methods when chemical treatments are involved.

Target Specificity

This product is not an antibody or affinity reagent and does not have antigen/epitope specificity in the immunochemical sense.

Assay specificity (general/literature)

• Chemical selectivity arises from the reactivity of 2′-deoxyribose under strong acid to yield aldehydic species that condense with diphenylamine, producing a blue chromophore. Ribose (RNA) reacts much less efficiently, providing preferential response to DNA.
• Certain carbohydrates and aldehyde-containing compounds can generate background signal; rigorous blanks and matrix-matched calibration mitigate this.

Item-specific claims of selectivity or cross-reactivity are not provided here; refer to the kit insert and validation data for performance characteristics.

자주 묻는 질문

What does BioReagent mean?

BioReagent indicates that the product has been processed and tested for biological and biochemical applications. Specifications may include endotoxin limits, microbial controls, sterility, low DNase/RNase/protease activity, and verified biological activity where relevant.

How should this product be stored?

Store at ?20 °C. Freezer storage is required to maintain the specified shelf life.