Blue High Sensitivity Double-Stranded DNA Quantification Kit differs from conventional absorbance-based assays. It can distinguish dsDNA, ssDNA and RNA, enabling selective detection of dsDNA (Figure 2). Compared with traditional DNA quantitation methods, this product features a wide detection range, high sensitivity and high specificity.
The Blue High Sensitivity dsDNA Quantitation Kit quantifies dsDNA ranging from 0.2 to 100 ng in a 200 μL reaction system (Figure 3). In addition, it minimizes interference from other contaminants and tolerates common impurities such as proteins, salts, organic solvents and detergents (Table 1). Moreover, the reagent is cell-impermeable, non-cytotoxic and non-mutagenic, making it safe for human use.
Contents & Storage:
| Component No. | Component Name | Appearance | 200 T | 1000 T | Storage |
| W1372296A | Blue High Sensitivity dsDNA Quantitation Solution | Liquid | 50 mL | 250 mL | 2-8 ℃. Store in the dark. |
| W1372296B | Blue High Sensitivity dsDNA Enhancer, 100× | Liquid | 0.5 mL | 2 ×1.25 mL | 2-8 ℃. Store in the dark. |
| W1372296C | dsDNA Standards. 0 ng/uL dsDNA | Liquid | 0.1 mL | 0.5 mL | 2-8 ℃. Store in the dark. |
| W1372296D | dsDNA Standards. 0.5 ng/uL dsDNA | Liquid | 0.1 mL | 0.5 mL | 2-8 ℃. Store in the dark. |
| W1372296E | dsDNA Standards. 1 ng/uL dsDNA | Liquid | 0.1 mL | 0.5 mL | 2-8 ℃. Store in the dark. |
| W1372296F | dsDNA Standards. 2 ng/uL dsDNA | Liquid | 0.1 mL | 0.5 mL | 2-8 ℃. Store in the dark. |
| W1372296G | dsDNA Standards. 4 ng/uL dsDNA | Liquid | 0.1 mL | 0.5 mL | 2-8 ℃. Store in the dark. |
| W1372296H | dsDNA Standards. 6 ng/uL dsDNA | Liquid | 0.1 mL | 0.5 mL | 2-8 ℃. Store in the dark. |
| W1372296I | dsDNA Standards. 8 ng/uL dsDNA | Liquid | 0.1 mL | 0.5 mL | 2-8 ℃. Store in the dark. |
| W1372296J | dsDNA Standards. 10 ng/uL dsDNA | Liquid | 0.1 mL | 0.5 mL | 2-8 ℃. Store in the dark. |
Storage Conditions
Store at 4 °C, protected from light. Expiry date is indicated on the outer package.
Product Specifications
Ex/Em: 485/530 nm (when bound to dsDNA)
Figure 1. Excitation and emission spectra of Blue under dsDNA-saturated conditions
Protocol:
1. For optimal results, use accurately calibrated pipettors and RNase-free tips, tubes and assay plates. It is recommended to set up 3 technical replicates for each DNA standard and unknown sample. If multiple 96-well plates are assayed, a standard curve should be prepared for each plate to minimize inter-plate variation.
2. Remove the kit from storage and equilibrate to room temperature before use. If Component B forms a precipitate, dissolve it in a 37 °C water bath. Vortex and centrifuge each component thoroughly to avoid unnecessary reagent loss.
3. Prepare 200 µL of Blue working solution per test sample. For one 96-well plate, transfer 200 µL of Component B into 20 mL of Component A, then vortex to prepare Blue working solution. For best performance, use the working solution within 1 hour. If the working solution is stored and reused within 24 h, a slight reduction in accuracy may occur. Precipitation may form in the working solution during storage; re-suspend by vortexing.
4. Add 200 µL of working solution into each well of a black 96-well plate. For reliable and accurate results, prepare 3 parallel technical replicates for each test sample and DNA standard. A properly sized multichannel pipettor may be used for this step. Black plates reduce fluorescence crosstalk between samples.
5. Add 10 µL of dsDNA standard or 1–20 µL of unknown sample into each well, and mix gently by pipetting.
6. Incubate the plate at room temperature, protected from light, for 5–10 min. For optimal results, read the plate immediately after incubation. Readings may be taken within 6 h, with a slight loss in precision.
7. Measure fluorescence intensity using a microplate reader with excitation at 485 nm and emission at 530 nm.
8. Generate a standard curve and calculate the DNA concentration of test samples (see Figure 2).
Note: The standard curve in Figure 3 is for reference only. Prepare your own standard curve using your experimental data to calculate sample concentrations.
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Figure 2. Relative fluorescence intensity of different nucleic acids detected using the Blue High Sensitivity dsDNA Quantitation Kit.
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Figure 3. Standard curve of calf thymus DNA generated with the Blue High Sensitivity dsDNA Quantitation Kit (Ex/Em 485/530 nm).
Experimental Considerations:
1. For DNA samples derived from plants or animals, calf thymus DNA is commonly used as the reference for preparing DNA standards. It is double‑stranded, highly polymerized, and features an even base composition (58% AT, 42% GC). If the fluorescence reading of a test sample exceeds the range of the standard curve, further dilution of the sample is required. To ensure consistent results, maintain equal loading volume in each well and avoid high concentrations of other contaminants.
2. The Blue High Sensitivity dsDNA Quantitation Kit quantifies dsDNA within the range of 0.2–100 ng. For samples where linear quantitation is not required, the detectable range of the kit can be extended to 200 ng. For samples with lower DNA levels, dilute the DNA standards with 1×TE buffer down to 0.02 ng/μL, then load 10 μL per well following the standard protocol.
3. Instrument settings may be optimized for different plate readers to achieve optimal linearity. Factors that can affect final linearity and relative fluorescence intensity include: (1) excitation and emission wavelengths and bandwidth; (2) cutoff filters; (3) gain/sensitivity settings; (4) pipetting accuracy; (5) microplate manufacturer.
4. Table 1 lists several common DNA contaminants, including salts, solvents, detergents and proteins.
Table 1. Effects of Common DNA Contaminants on the Blue High Sensitivity dsDNA Quantitation Kit
Compound | Stock Concentration | Final Concentration (200µL) | Result | Ammonium acetate | 100 mM | 5 mM | Pass | Sodium acetate | 600 mM | 30 mM | Pass | Sodium chloride | 200 mM | 10 mM | Pass | Magnesium chloride | 25 mM | 1.25 mM | Pass | Phenol | 2 % | 0.10 % | Pass | Ethanol | 10 % | 0.5 % | Pass | Chloroform | 2 % | 0.1 % | Pass | Sodium dodecyl sulfate (SDS) | 0.2 % | 0.01 % | Pass | Triton X-100 | 0.2 % | 0.01 % | Pass | Bovine serum albumin (BSA) | 200 mg/mL | 10 mg/mL | Pass* | dNTPs** | 2 mM | 100 µM | Pass | Polyethylene glycol | 40 % | 2 % | / | Agarose | 2 % | 0.1 % | /
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Note: Standard curves were generated using triplicate dsDNA samples under the conditions with the indicated contaminants at the stock concentrations listed above. “Pass” means the variation of experimental results is less than 20% compared with the system without contaminants. Fluorescence intensity of all samples was measured on a Molecular Devices Gemini XS microplate reader with excitation at 485 nm and emission at 530 nm. “Pass*” indicates a slight shift in the standard curve obtained under this condition. “dNTPs**” refers to a mixture of dATP, dCTP, dGTP and dTTP.
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Precautions:
1. Briefly centrifuge the product to collect contents at the bottom of the tube before use.
2. Fluorescent dyes are susceptible to photobleaching. Protect the reagent from light to slow down photobleaching.
3. For your safety and health, wear a lab coat and disposable gloves during operation.
