dsDNA BR Assay Kit——StarLighter

Description

Laboratories quantifying double-stranded DNA may encounter interference when using traditional UV absorbance at 260 nm. RNA, proteins, salts, detergents, and other substances can affect absorbance-based measurements, particularly when samples must be measured across a broad concentration range.

The StarLighter dsDNA BR Assay Kit provides a fluorescence-based solution for broad-range dsDNA quantification. The assay is highly selective for double-stranded DNA over RNA and supports initial dsDNA concentrations from 0.2 ng/µL to 1,000 ng/µL, depending on the sample volume. Its total assay range is 2–1,000 ng.

This dsDNA broad range assay kit requires only 1–20 µL of sample in a final assay volume of 200 µL. It can be used with either a Qubit fluorometer or a fluorescence microplate reader, allowing laboratories to perform individual tube measurements or 96-well fluorescence-based dsDNA quantitation.

The assay is performed at room temperature after a 2-minute incubation protected from light. 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

• Broad-range dsDNA quantification: The fluorescent dsDNA quantitation kit covers a total assay range of 2–1,000 ng.
• Selective dsDNA measurement: The assay is highly selective for double-stranded DNA over RNA.
• Flexible sample input: The assay accepts 1–20 µL of sample in a final volume of 200 µL.
• Two instrument formats: The kit can be used with a Qubit fluorometer or a fluorescence microplate reader.
• 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 after incubation when protected from light.
• Contaminant tolerance: The assay was evaluated with common laboratory contaminants, including salts, solvents, detergents, protein, agarose, PEG, and dNTPs.
• Two kit sizes: The dsDNA BR assay kit is available in 100-reaction and 1,000-reaction formats.

Contaminant Tolerance

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

Application

Broad-Range 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 using thin-wall, clear 0.5 mL PCR tubes.
3. Prepare fresh Working Solution by combining dsDNA BR Fluorescent Reagent QF and dsDNA BR Buffer QB at a QF:QB ratio of 1:199.
4. Do not prepare or mix the Working Solution in a glass container.
5. Add 190 µL of 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 QS0 to the first standard tube and 10 µL of 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. Vortex vigorously for 3–5 seconds without creating bubbles.
10. Incubate at room temperature for 2 minutes while protected from light.
11. On a Qubit 4 Fluorometer, select dsDNA and then dsDNA Broad Range.
12. Read the two standards in the correct order before reading the samples.

A new calibration is strongly recommended for every assay run. Calibration curves generated using other kits must not be used.

Broad-Range dsDNA Quantification with a Fluorescence Microplate Reader

1. Equilibrate all kit components to room temperature and mix thoroughly by vortexing.
2. Prepare fresh Working Solution by combining QF and QB at a QF:QB ratio of 1:199.
3. Use a black-wall, clear-bottom 96-well microplate to minimize cross-talk.
4. Add 190 µL of Working Solution to each standard well.
5. Add 180–199 µL of Working Solution to each sample well, depending on the sample volume.
6. Serially dilute Standard QS1 to prepare standards ranging from 0 to 100 ng/µL.
7. Add 10 µL of each standard to the designated standard wells.
8. Add 1–20 µL of each sample to the corresponding wells. The final volume must be exactly 200 µL per well.
9. Vortex the plate briefly for 10–15 seconds.
10. Incubate the plate for 2 minutes at room temperature while protected from light.
11. Set the microplate reader to 485 nm excitation and 530 nm emission.
12. Read the standards and samples, generate a standard curve, and calculate the concentrations of the unknown samples.

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

FAQ