Extended Range
The upper detection limit can be extended to 4000 ng/µL by adding 1 µL of a 4000 ng/µL sample to 199 µL of working reagent.
Note: There is some loss of linearity with this assay when adding more than 2000 ng total mass per assay tube.
Kit Contents
Three assay sizes are available. The volume of components in each kit are sufficient for 1000, 250 and 50 (evaluation size) assays respectively. Kit components are shown below. Safety data sheets are available at denovix.com/sds.
Table 1: dsDNA Broad Range Assay Kit Contents| Component | 1000 | 250 | EVAL |
|---|---|---|---|
| DeNovix dsDNA Broad Range Dye (100x) | 2 x 1 mL | 0.5 mL | 100 µL |
| DeNovix dsDNA Broad Range Buffer | 250 mL | 50 mL | 10 mL |
| DeNovix dsDNA Broad Range Enhancer (100x) | 2 x 1 mL | 0.5 mL | 100 µL |
| 200 ng/µL dsDNA Standard (calf thymus) | 2 mL | 1 mL | 0.5 mL |
| 0 ng/µL dsDNA Standard | 2 mL | 1 mL | 0.5 mL |
Instrument Compatibility
The spectral properties of the dye are excitation /emission of 350/460 nm in the presence of dsDNA as shown in Figure 1 below.
The kit is compatible with fluorescence microplate readers and fluorometers with the appropriate excitation sources and emission detectors.
Specific instructions using the 2 Point Standard Assay with DeNovix DS-11 FX, FX module or the QFX fluorometer are included in Technical Note 142.
Assay Considerations
Calf thymus DNA is provided as the reference standard as it is double-stranded, highly polymerized and approximately 58% AT (42% GC). It may be preferable to use an alternatively dsDNA standard more similar (e.g., similar size, linear vs. circular) to the unknown samples of interest. For bacterial DNA, consider using a species-specific standard, as the GC content varies widely depending on the species.
Although many instruments, including DeNovix DS-11 Fluorometers, offer the option to use previously saved values, it is recommended that a new standard curve be generated at the time of the assay for optimal results.
Assay Linearity and Detection Limits
Fluorescent quantification specifications are often expressed in a variety of conventions. The full detection range (including the extended range) of this assay can be expressed in the following specifications:
Table 2: Broad Range Assay Linearity and Detection Limits| Specification | Range |
|---|---|
| Absolute mass per assay tube | 2 – 2000 ng per 200 µL |
| Concentration in sample stock tube | 100 pg/µL – 4000 ng/µL |
Reagent Storage
The kit is stable for 12 months from ship date when stored as recommended.
Table 3: Broad Range Assay Reagant Storage| Component | Protect from Light | Temperature |
|---|---|---|
| DeNovix dsDNA Broad Range Dye (100x)* | Yes | 4°C - Room Temperature |
| DeNovix dsDNA Broad Range Buffer | Optional | 4°C - Room Temperature |
| DeNovix dsDNA Broad Range Enhancer (100x) | Optional | 4°C - Room Temperature |
| dsDNA Standards | Yes | 4°C |
* Note: The DeNovix dsDNA Broad Range dye is provided in DMSO, which may freeze if stored at 4°C.
Best Practices
Assay Protocol
- Allow all solutions to equilibrate to room temperature before use.
- Vortex, then centrifuge vials briefly before opening to minimize reagent loss on the cap.
- Prepare working solution by mixing the dye and enhancer each with the assay buffer in a 1:100 ratio, e.g. 100 uL dye and 100 uL enhancer into 10 mL buffer.
- Scale volumes as needed to make enough volume to aliquot 190 µL of the mixture per standard and unknown to be measured.
- For each standard or unknown sample, add 190 µL of the working solution to a labeled tube or micro well. Adjust volume when adding more or less than 10 µL of the unknown sample.
- Add 10 µL of the 0 ng/µL and 200 ng/µL standards and 1 – 20 µL of unknown DNA samples to the respective tubes and mix well.
- Incubate standards and samples at room temperature for 5 minutes.
- Generate the standard curve and then measure the samples using the proper excitation source and emission filters.
Recommended Sample Volume
These recommendations ensure that sample concentrations are within the total mass detection limits of the assay. Total assay volume should remain 200 µL. Adjust working solution volume accordingly.
| Initial Sample Concentration | Recommended Sample Volume |
|---|---|
| 0.2 – 200 ng/µL | 10 µL |
| 0.1 – 2 ng/µL | 20 µL |
| 200 – 1000 ng/µL | 2 µL |
| 1000 – 4000 ng/µL | 1 µL |
Standard Dilutions
Preparing diluted standards is not required when using the 2 point assay option supplied. For the DeNovix User Defined Standards option, or for use on microplate readers, prepare DNA standards by serial dilution of the 200 ng/µL standard provided in 1X TE buffer (10 mM Tris pH 7-8, 1 mM EDTA).
Table 4: Broad Range Assay Standard Dilutions| Standard | DNA | TE |
|---|---|---|
| 200 ng/µL | 275 µL of 200 ng/µL stock tube | None |
| 150 ng/µL | 75 µL of 200 ng/µL standard | 25 µL |
| 100 ng/µL | 100 µL of 200 ng/µL standard | 100 µL |
| 25 ng/µL | 50 µL of 100 ng/µL standard | 150 µL |
| 12.5 ng/µL | 100 µL of 25 ng/µL standard | 100 µL |
| 6.25 ng/µL | 100 µL of 12.5 ng/µL standard | 100 µL |
| 2 ng/µL | 32 µL of 6.25 ng/µL standard | 68 µL |
| 0 ng/µL | None | 100 µL |
Data Analysis
Sample concentrations are automatically calculated when using a DeNovix DS-11 FX or QFX Fluorometer.
For all other instruments, follow the instructions below:
- Generate a standard curve to determine the unknown DNA concentration.
- Average replicates’ values for each sample and subtract the average zero DNA value from each data point.
- Plot the fluorescence RFU values for the DNA standards on the y-axis and ng/well DNA on the x-axis, and fit a trend line (Figure 2) through these points to generate a standard curve with a y-intercept = 0.
- Use the equation for the trend line to calculate the amount of unknown DNA in each well (y = fluorescence and x = ng DNA per well or tube).
Solvent Compatibility
Table 5. The tolerance of the dye in the presence of common solvents while maintaining linearity.| Compound | Final maximum concentration in assay (200 µL) |
|---|---|
| Ammonium Acetate | 5 mM |
| Sodium Chloride | 50 mM |
| Ethanol | 0.5 % |
| Phenol | 0.1 % |
| SDS | 0.0 1% |
| dNTPs* | 100 µM |
Troubleshooting
DeNovix Assays
If the Broad Range Assay does not cover the concentration range of your samples, consider using an alternate DeNovix dsDNA Assay Kit.
For comparison, the standard detection ranges of the three assays are seen in Table 6.
Table 6: DeNovix Assays
Assay Detection Ranges| DeNovix dsDNA Assay | Range |
|---|---|
| Broad Range | 0.1 – 2000 ng/µL (extended range to 4000 ng/uL) |
| High Sensitivity | 10 pg/µL – 250 ng/µL (extended range down to 5 pg/uL) |
| Ultra High Sensitivity | 0.5 – 300 pg/µL |
01-MAR-2023
