StarLighter dsDNA High Sensitivity Detection Kit

The StarLighter dsDNA HS (High Sensitivity) Assay Kit is a simple, rapid, sensitive, and highly accurate fluorescent quantification kit for double-stranded DNA (dsDNA). This kit includes fluorescent detection reagents, standards, and buffer. It is highly selective for dsDNA, minimizes RNA interference, and exhibits excellent linearity for dsDNA samples between 0.1 and 100 ng. Compared to traditional UV detection (A260) methods, the kit is largely immune to interference from other substances, such as RNA and protein, enabling more precise DNA quantification and improving experimental accuracy. This kit is easy to use and can be used with a fluorescence microplate reader or Qubit fluorometer.

Product components

Transportation and storage conditions

Store at 2 ~ 8℃ and transport with ice packs.

Features

High sensitivity: can accurately quantify dsDNA samples from 0.1 to 100 ng/µL;

Strong specificity: only binds to dsDNA and is not affected by RNA, protein, etc.

Good tolerance: It can tolerate higher concentrations of salt, urea, ethanol, detergents, proteins or agarose, and can directly quantify PCR amplification products;

The operation is simple: dilute the fluorescent reagent with buffer to form a working solution, then add the dsDNA sample to be tested and start the test. This operation can be performed at room temperature.

Precautions

1. Each component of the kit must be mixed thoroughly before each use.
2. All fluorescent dyes have quenching problems. Please avoid light to slow down fluorescence quenching.
3. When using the Qubit Fluorometer for detection, recalibration with the standard reagents must be performed to ensure the accuracy of the results.
4. When using the Qubit Fluorometer, use 5 mL thin-walled, clear assay tubes.
5. If the concentration of the sample to be tested is higher than 100 ng/µL, please dilute it appropriately before testing.

Experimental operation process

1. use Qubit Fluorometer dsDNA Quantitative detection and analysis
   • Before use, be sure to equilibrate all components in the kit to room temperature and mix thoroughly;
   • Prepare the assay working solution. Prepare the assay working solution at a volume ratio of 1:199 QF:QB. Prepare immediately before use. For example, if you are testing 8 DNA samples and 2 standards, allowing for aliquot losses, we recommend preparing a working solution for 11 samples: 11 µL QF + 2189 µL QB buffer. Vortex to mix.
   • Add 190 µL of freshly prepared working solution to each standard tube, and add 180 to 199 µL of freshly prepared working solution to each sample tube (depending on the volume of sample added, 1 to 20 µL can be added to each sample to bring the total volume to 200 µL).
   • Add 10 µL of DNA standard QS0 and DNA standard QS1 to the corresponding standard tubes. Add a certain volume (1 to 20 µL) of the dsDNA sample to be tested to the sample tube to make the total volume in the tube reach 200 µL. Vortex mix for 2 to 3 seconds.
   • Incubate the standard tube and sample tube at room temperature in the dark for 2 minutes;
   • According to the operating instructions of the Qubit Fluorometer, select the dsDNA High Sensitivity assay program to measure the fluorescence signal value.

2. Quantitative detection of dsDNA using a fluorescence microplate reader
   • Before use, be sure to equilibrate all components in the kit to room temperature and mix thoroughly;
   • Prepare the assay working solution. Prepare the assay working solution at a volume ratio of 1:199 QF:QB. Prepare the solution immediately before use. For example, if testing five DNA samples, five standards are also required. These standards can be serially diluted from standard S1, with a concentration range of 0 to 10 ng/µL. Therefore, theoretically, a working solution for ten samples is required. Allowing for aliquot losses, it is recommended to prepare a working solution for eleven samples: 11 µL QF + 2189 µL QB buffer. Vortex to mix.
   • Add freshly prepared assay working solution to a 96-well microtiter plate. Add 190 µL of working solution to the standard wells and 180-199 µL of working solution to the DNA sample wells.

Note ：It is recommended to use black ELISA plates, which can effectively reduce the interference of fluorescent signals between reaction wells.

• Take the DNA standard QS1 in the kit and dilute it according to the concentration gradient to prepare a series of diluted dsDNA standards (the concentration range should be 0 ~ 10 ng/µL);
• Add 10 µL of a dsDNA standard of varying concentrations to the standard wells of a 96-well microtiter plate. Add 1 to 20 µL of the dsDNA sample to be tested to the sample wells of the 96-well microtiter plate to a total volume of 200 µL. Vortex mix for 2 to 3 seconds.
• Incubate the ELISA plate in the dark at room temperature for 2 min.
• The fluorescence signal value was detected using a fluorescence microplate reader, and the appropriate detection wavelength was selected: the excitation wavelength (Ex) was set to 485 nm, and the emission wavelength (Em) was set to 530 nm;
• The measured fluorescence signal values of the dsDNA standards correspond to their concentrations and a standard curve is drawn. The measured fluorescence signal values of dsDNA samples of unknown concentration are substituted into the standard curve to calculate the concentration of the dsDNA sample.

Appendix: Impact of Contaminants on Quantitative Detection Kits