dsDNA HS Assay Kit——StarLighter

Description

Low-concentration double-stranded DNA samples can be difficult to quantify using traditional UV A260 measurements. The StarLighter dsDNA HS Assay Kit provides a high-sensitivity, fluorescence-based method for selective quantification of double-stranded DNA. The assay detects initial dsDNA concentrations from 5 pg/µL to 120 ng/µL, depending on sample volume, with a total assay range of 0.1–120 ng.

The kit contains a fluorescent reagent, assay buffer, and two dsDNA standards. It provides high selectivity for dsDNA over RNA and tolerates specified concentrations of common laboratory contaminants, including salts, free nucleotides, solvents, detergents, and proteins.

The protocol supports two measurement formats. Samples can be measured individually using a Qubit fluorometer and 0.5 mL thin-walled clear analysis tubes, or processed in a black 96-well microplate using a fluorescence microplate reader. Each assay accepts 1–20 µL of sample in a final reaction volume of 200 µL.

The working solution is prepared by diluting dsDNA HS Fluorescent Reagent QF into dsDNA HS Buffer QB at a 1:199 ratio. After mixing, the reaction requires a 2-minute incubation at room temperature while protected from light. The fluorescent signal remains stable for up to 3 hours after incubation when protected from light.

The StarLighter dsDNA HS Assay Kit is available in 20-reaction and 500-reaction formats. This product is for research use only and is not intended for diagnostic or therapeutic procedures.

Specification

Catalog Numbers and Kit Components

Required Materials Not Supplied

• Fluorometer and 0.5 mL thin-walled clear analysis tubes, or a fluorescence microplate reader and black 96-well microplates
• Vortex mixer
• Pipettes and sterile, nuclease-free pipette tips
• Nuclease-free water or TE buffer for sample dilution, if required

Features

• High-sensitivity dsDNA quantification: Detects initial dsDNA concentrations from 5 pg/µL to 120 ng/µL, depending on sample volume.
• Defined total assay range: Measures 0.1–120 ng of dsDNA per assay.
• Selective fluorescence measurement: Provides high selectivity for double-stranded DNA over RNA.
• Low sample requirement: Supports sample volumes from 1 to 20 µL.
• Two measurement formats: The protocol can be performed using a Qubit fluorometer or a fluorescence microplate reader.
• 96-well assay format: Supports fluorescence-based dsDNA quantification in black-wall, clear-bottom 96-well microplates.
• Short incubation: Requires a 2-minute incubation at room temperature while protected from light.
• Stable post-incubation signal: The fluorescent signal remains stable for up to 3 hours when protected from light.
• Two supplied standards: Includes QS0 at 0 ng/µL and QS1 at 10 ng/µL.
• Two package sizes: Available as FS-T1001-S for 20 reactions and FS-T1002 for 500 reactions.

Documented Contaminant Tolerance

The following table lists the contaminant concentrations evaluated in the assay. Results reported as “OK” usually produced less than 10% perturbation under the stated conditions.

Application

The StarLighter dsDNA HS Assay Kit is intended for fluorescence-based quantification of double-stranded DNA using either a Qubit fluorometer or a fluorescence microplate reader.

Qubit Fluorometer Assay Format

1. Equilibrate all kit components to room temperature.
2. Prepare the working solution by combining dsDNA HS Fluorescent Reagent QF and dsDNA HS Buffer QB at a 1:199 ratio.
3. Use thin-walled, clear 0.5 mL PCR tubes.
4. Add 190 µL of working solution and 10 µL of standard to each standard tube.
5. Add 180–199 µL of working solution and 1–20 µL of sample to each sample tube.
6. Ensure that the final volume in every tube is 200 µL.
7. Vortex for 3–5 seconds without creating bubbles.
8. Incubate for 2 minutes at room temperature while protected from light.
9. Read the standards and samples using the applicable instrument procedure.

Calibration requires two standards. Standard QS0 is used at 0 ng/µL, and Standard QS1 is used at 10 ng/µL. A new calibration is strongly recommended for every new assay run, and calibration curves from other kits must not be used.

Fluorescence Microplate Reader Assay Format

1. Equilibrate all kit components to room temperature and mix thoroughly by vortexing.
2. Prepare the working solution using a 1:199 ratio of QF fluorescent reagent to QB buffer.
3. Dispense 190 µL of working solution into standard wells.
4. Dispense 180–199 µL of working solution into sample wells according to the sample volume.
5. Prepare standards from 0 to 10 ng/µL by serially diluting Standard QS1.
6. Add 10 µL of each standard and 1–20 µL of each sample to the designated wells.
7. Ensure that the final volume in each well is exactly 200 µL.
8. Mix the plate for 10–15 seconds.
9. Incubate for 2 minutes at room temperature while protected from light.
10. Set the fluorescence microplate reader to 485 nm excitation and 530 nm emission.
11. Read the standards and samples, generate a standard curve, and calculate the concentrations of the unknown samples.

Black-wall, clear-bottom microplates are specified for the 96-well format to minimize cross-talk. Samples exceeding the linear range should be diluted and measured again.

FAQ