1X dsDNA HS Assay Kit——StarLighter

Description

Laboratories quantifying low-concentration double-stranded DNA may encounter measurement interference when using traditional UV absorbance at 260 nm. RNA, proteins, salts, free nucleotides, solvents, and detergents can affect DNA measurements, while preparing a working solution before every assay adds another handling step.

The StarLighter 1× dsDNA HS Assay Kit provides a ready-to-use fluorescence-based method for high-sensitivity double-stranded DNA quantification. The supplied 1× dsDNA HS Working Solution does not require dilution or preparation before use. Add the required volume of sample to the working solution, incubate for 2 minutes at room temperature while protected from light, and measure the fluorescence.

This ready-to-use 1X dsDNA HS assay kit is highly selective for dsDNA over RNA. Depending on sample volume, it supports initial dsDNA concentrations from 10 pg/µL to 100 ng/µL and a total assay range of 0.2–100 ng. Each assay uses 1–20 µL of sample in a final volume of 200 µL.

The fluorescent dsDNA quantitation kit is compatible with Qubit 2, Qubit 3, Qubit 4, and Qubit Flex fluorometers. It can also be used with a fluorescence microplate reader at 485 nm excitation and 530 nm emission. When protected from light, the fluorescent signal remains stable for up to 3 hours after incubation.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

Specification

Available Sizes and Kit Components

Features

• Ready-to-use formulation: The supplied 1× dsDNA HS Working Solution can be used directly without preparing a diluted working solution.
• High-sensitivity dsDNA quantification: The assay covers a total DNA range of 0.2–100 ng.
• Selective dsDNA measurement: The assay is highly selective for double-stranded DNA over RNA.
• Flexible sample input: Each assay accepts 1–20 µL of sample in a final volume of 200 µL.
• Short incubation: Fluorescent signal development requires 2 minutes at room temperature while protected from light.
• Stable reading window: The signal remains stable for up to 3 hours when samples are protected from light.
• Multiple instrument formats: The kit can be used with Qubit fluorometers or a fluorescence microplate reader.
• Contaminant tolerance: The assay was evaluated in the presence of salts, solvents, detergents, protein, ssDNA, RNA, PEG, and dNTPs.

Contaminant Tolerance

DNA standards were tested in the presence or absence of the contaminants listed below. An “OK” result usually represents less than 10% perturbation at the indicated final assay concentration.

Application

High-Sensitivity dsDNA Quantification with a Qubit Fluorometer

1. Equilibrate all kit components to room temperature before use.
2. Prepare two standard tubes and the required number of sample tubes.
3. Use only thin-wall, clear 0.5 mL PCR tubes.
4. Label the tube lids. Do not label the sides of the tubes because this may interfere with sample reading.
5. Add 190 µL of ready-to-use 1× dsDNA HS Working Solution to each standard tube.
6. Add 180–199 µL of Working Solution to each sample tube, depending on the sample volume.
7. Add 10 µL of Standard QS0 to the first standard tube and 10 µL of Standard QS1 to the second standard tube.
8. Add 1–20 µL of sample to the appropriate sample tube. The final volume must be 200 µL.
9. When adding 1–2 µL of sample, use a 2 µL pipette for best results.
10. Vortex each tube vigorously for 3–5 seconds.
11. Incubate all tubes for 2 minutes at room temperature while protected from light.
12. On the Qubit 4 Fluorometer, select dsDNA and then 1× dsDNA High Sensitivity.
13. Read Standard QS0 followed by Standard QS1, then proceed to the samples.
14. Select the sample volume and output concentration units before reading each sample.

Do not hold assay tubes in your hand before reading because warming the solution may change the fluorescence measurement. Do not use calibration curves generated with other kits.

High-Sensitivity dsDNA Quantification with a Fluorescence Microplate Reader

1. Equilibrate all kit components to room temperature and mix thoroughly by vortexing.
2. Use a black-wall, clear-bottom 96-well microplate to minimize cross-talk.
3. Add 190 µL of ready-to-use 1× dsDNA HS Working Solution to each standard well.
4. Add 180–199 µL of Working Solution to each sample well, depending on the sample volume.
5. Serially dilute Standard QS1 to prepare standards ranging from 0 to 10 ng/µL.
6. Add 10 µL of each standard to the designated standard wells.
7. Add 1–20 µL of each sample to the corresponding sample wells.
8. Ensure that the final volume in every well is exactly 200 µL.
9. Vortex the plate briefly for 10–15 seconds.
10. Incubate the microplate for 2 minutes at room temperature while protected from light.
11. Set the fluorescence microplate reader to 485 nm excitation and 530 nm emission.
12. Read the fluorescence of the standards and samples.
13. Generate a standard curve from the serially diluted standards and calculate the concentrations of the unknown samples.

If a sample concentration exceeds the linear range, dilute the sample appropriately and repeat the assay.

FAQ