Extended Range
The lower detection limit can be extended down to 5 pg/µL sample by adding 20 µL of the 5 pg/µL sample to 180 µL of the working reagent.
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.
| Component | 1000 | 250 | EVAL |
|---|---|---|---|
| DeNovix dsDNA High Sensitivity Dye (100x) | 2 x 1 mL | 0.5 mL | 100 µL |
| DeNovix dsDNA High Sensitivity Buffer | 200 mL | 50 mL | 10 mL |
| 25 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 |
The dye is a potentially harmful chemical. Exercise universal laboratory safety precautions when handling the dye, and dispose of the dye as hazardous chemical waste according to your local regulations.
Instrument Compatibility
The DeNovix dsDNA High Sensitivity Quantitation Assay is designed for use with fluorometers or fluorescence plate readers equipped with excitation and emission filters for detecting green fluorescence. The unique spectral properties of the kit dye make it especially well-suited for use with instruments with blue LED excitation sources (Figure 1).
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 144.
Assay Considerations
Calf thymus DNA is provided as the reference standard as it is double-stranded, highly polymerized and is approximately 58% AT (42% GC). At times it may be preferable to use a dsDNA standard similar to the unknown samples (eg. similar in size, linear vs circular). For bacterial DNA, consider using a species-specific standard, as the GC content varies widely depending on the species.
Although many instruments, including the DeNovix DS-11 FX and QFX 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 1: High Range Assay Linearity and Detection Limits| Specification | Range |
|---|---|
| Absolute mass per assay tube | 100 pg – 250 ng per 200 µL |
| Concentration in sample stock tube | 5 pg/µL – 250 ng/µL |
Reagent Storage
The kit is stable for 12 months from ship date when stored as recommended.
Table 2: High Sensitivity AssayReagent Storage| Component | Protect from Light | Temperature |
|---|---|---|
| DeNovix dsDNA High Sensitivity Dye (100x) | Yes | 4oC - Room Temperature |
| DeNovix dsDNA High Sensitivity Buffer | Optional | 4oC - Room Temperature |
| dsDNA Standards | Yes | 4oC |
Best Practices
It is important to pay careful attention to pipetting accuracy and overall sample handling techniques when quantitating picogram amounts of dsDNA.
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 with the assay buffer in a 1:100 ratio, e.g. 100 uL dye into 10 mL buffer. Scale volumes as needed to make enough volume to aliquot 190 µL of the mixture for each standard and unknown. Mix well before use.
- For each standard or unknown sample, add 190 µL of the working solution per tube or micro well. Adjust volume when adding more or less than 10 µL of the unknown sample.
- Add 10 µL of each standard and between 1 – 20 µL of the unknown DNA sample to the assay tube or micro well and mix well.
- Incubate standards and samples at room temperature for 5 minutes. The assay is stable for 4 hours at room temperature.
- Generate the standard curve and then measure the samples using the proper excitation source and emission filters. The DeNovix DS-11 FX or QFX software automatically utilizes the correct excitation and emission setup.
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 |
|---|---|
| 10 pg/µL – 25 ng/µL | 10 µL |
| 5 – 100 pg/µL | 20 µL |
| 25 – 125 ng/µL | 2 µL |
| 125 – 250 ng/µL | 1 µL |
Standard Dilutions
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 a set of DNA standards by serial dilution of the 25 ng/µL standard in 1X TE buffer (10 mM Tris pH 7-8, 1 mM EDTA) as shown in the table below.
| Standard | DNA | TE |
|---|---|---|
| 25 ng/µL | 100 µL of 25 ng/µL stock tube | None |
| 10 ng/µL | 40 µL of 25 ng/µL standard | 60 µL |
| 2.5 ng/µL | 25 µL of 10 ng/µL standard | 75 µL |
| 1 ng/µL | 40 µL of 2.5 ng/µL standard | 60 µL |
| 0.25 ng/µL | 25 µL of 1 ng/µL standard | 75 µL |
| 0.1 ng/µL | 40 µL of 0.25 ng/µL standard | 60 µL |
| 0.03 ng/µL | 30 µL of 0.1 ng/µL standard | 70 µL |
| 0 ng/µL | 100 µL of 0 ng/µL stock tube | None |
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 the triplicate 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).
Troubleshooting
Appendix: Solvent Compatibility
If the Broad Range Assay does not cover the concentration range of your samples, consider using an alternate DeNovix dsDNA Assay Kit.
| Compound | Maximum concentration in 200 µL assay | Signal Decrease (%) |
|---|---|---|
| Ammonium Acetate | 50 mM | 0.14 |
| Sodium Chloride | 50 mM | 0.14 |
| Magnesium Chloride | 5 mM | 0.16 |
| Sodium Acetate | 30 mM | 0.11 |
| Ethanol | 0.01 | 0.21 |
| Phenol | 0.001 | 0.11 |
| Chloroform | 0.01 | 0.34 |
| SDS | 0.0001 | 0.31 |
| SDS | 1.0E-5 | 0.09 |
| Trtion X-100 | 1.0E-5 | 0.2 |
| dNTPs | 100 µM | 0.11 |
DNA standards assayed in the absence or presence of contaminants listed at the concentrations above. A result of OK indicates that there was <20% change in the signal in the absence of the contaminant. *Mix of dATP, dCTP, dGTP, dTTP.
DeNovix Assays
The DeNovix dsDNA Broad Range and Ultra High Sensitivity Assays are more tolerant of some contaminants compared to the High Sensitivity Assay reagents. If the High Sensitivity Assay is not compatible with your dsDNA extraction procedure, consider using an alternate DeNovix dsDNA Assay Kit. For comparison, the standard detection ranges of the three assays are as follows:
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
