Introduction
The 1X dsDNA High Sensitivity (HS) Assay Kit for Qubit is a fluorescence-based quantification tool used to accurately measure low concentrations of double-stranded DNA (dsDNA). As molecular biology, next-generation sequencing (NGS), and single-cell genomics evolve, precise DNA quantification becomes indispensable. This kit is optimized for dsDNA concentrations ranging from 10 pg/μL to 100 ng/μL, enabling high-accuracy workflows with minimal sample input.
As outlined in the National Center for Biotechnology Information, traditional UV absorbance methods like NanoDrop™ suffer from RNA/protein contamination interference, while Qubit assays are highly specific for dsDNA.
How the 1X dsDNA HS Assay Kit Works
The kit contains a proprietary fluorescent dye that selectively binds to dsDNA, emitting a signal only upon binding. This eliminates quantification errors caused by single-stranded DNA, RNA, free nucleotides, or proteins. Fluorescence is detected using the Qubit fluorometer, which is calibrated with provided standards.
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Excitation: ~480 nm
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Emission: ~500–520 nm
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Linear range: 10 pg/μL – 100 ng/μL
According to NIH.gov, this method outperforms spectrophotometric absorbance in sensitivity and specificity, especially in low-yield or degraded DNA.
Step-by-Step Protocol for Qubit Quantification
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Dilute the Reagents
Mix dye with buffer (supplied) to create the working solution. -
Prepare Calibration Standards
Use the provided DNA standards to calibrate the Qubit device. -
Sample Preparation
Mix 1–20 µL of DNA sample with the working solution in a Qubit assay tube. -
Incubation
Let the mixture incubate at room temperature for 2 minutes (or as per CDC laboratory recommendations). -
Measurement
Insert the tube into the Qubit fluorometer and record fluorescence.
Detailed protocols are available at Yale’s Molecular Biology Core Facility and UCLA’s Genomics Center.
Applications in Modern Molecular Biology
1. Library Preparation for NGS
Accurate DNA input is critical for successful NGS workflows. NCBI guidelines on sequencing prep emphasize the importance of low-input, high-fidelity quantification tools.
2. Quantifying Fragmented DNA
Used in cell-free DNA (cfDNA) and ancient DNA studies. Refer to studies from Harvard Medical School and University of Washington Genome Sciences.
3. Single-Cell Genomics
In workflows described by the Broad Institute, precise quantification is critical to prevent amplification bias in scDNA-seq.
4. Gene Editing Quality Control
CRISPR workflows depend on accurate template DNA concentrations. See NIH’s CRISPR resources.
5. qPCR and ChIP-seq
Sample input normalization is essential in quantitative PCR (qPCR) and chromatin immunoprecipitation sequencing (ChIP-seq). Learn more at Encode Project and NIH’s Epigenomics Program.
Comparison with Other DNA Quantification Methods
| Method | Sensitivity | Specificity | Contaminant Interference |
|---|---|---|---|
| NanoDrop (UV) | Low | Non-selective | High |
| Agarose Gel | Medium | Low | High |
| Qubit 1X dsDNA HS Kit | High | High | Low |
Refer to the comparative evaluation published by NCBI and FDA research on nucleic acid testing.
Storage, Shelf Life, and Stability
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Store at 2°C–8°C
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Protect from light
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Shelf life: up to 12 months
Refer to FDA assay reagent guidelines and USDA lab standards.
Common Troubleshooting Scenarios
| Issue | Possible Cause | Solution |
|---|---|---|
| Low signal | Expired reagents or incorrect dilution | Use fresh working solution |
| High background fluorescence | Contamination with ssDNA/RNA | Use RNase-treated samples, ensure purity |
| Inconsistent results between runs | Variability in pipetting | Use calibrated pipettes, maintain technique |
More solutions can be found in Johns Hopkins University troubleshooting guides and University of Michigan’s Core Facilities.
Frequently Asked Questions (FAQs)
Q1: Can I reuse the Qubit dye or working solution?
No, the working solution must be freshly prepared. Refer to NIH’s best lab practices.
Q2: Can this assay be used with RNA samples?
No, the dye is highly specific for dsDNA. For RNA quantification, use the Qubit RNA HS kit, as recommended by CDC biosafety labs.
Q3: Is it compatible with degraded DNA?
Yes, as long as it is double-stranded. Studies by University of California, Davis support its use in forensic and cfDNA samples.
Why Use Qubit Over Spectrophotometry?
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Spectrophotometers like NanoDrop measure all nucleic acids at 260 nm, leading to false high readings due to RNA and nucleotide contamination.
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The Qubit system only detects dsDNA using dye-based fluorometry, which improves accuracy, especially in low-concentration samples (see NSF-funded studies).
Regulatory and Compliance
This assay kit is used in CLIA-certified and CAP-accredited labs as outlined by CMS.gov and CAP.org. It adheres to ISO 13485:2016 quality standards and is suitable for GxP environments.
Conclusion
The 1X dsDNA HS Assay Kit for Qubit is a gold-standard solution for high-sensitivity dsDNA quantification. It ensures accuracy, minimizes error, and enables reliable data for NGS, PCR, and molecular diagnostics. With its ability to work in low-volume, high-precision scenarios, it remains an indispensable reagent for modern labs, aligning with FDA, NIH, and academic best practices.
For more detailed usage, refer to official documentation from: