USER GUIDE

Qubit™ dsDNA HS Assay Kit

Catalog Numbers Q32851, Q32854
Pub. No. MAN0002326 Rev. C.0

WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,
clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support.

Product description
™
The Qubit dsDNA HS (High Sensitivity) Assay Kits make DNA quantitation easy and accurate. The assay is highly selective for double-
stranded DNA (dsDNA) over RNA (see “Assay selectivity” on page 2). Depending on sample volume, the assay is accurate for initial
sample concentrations from 5 pg/µL to 120 ng/µL providing an assay range of 0.1–120 ng. Common contaminants such as salts, free
nucleotides, solvents, detergents, or protein are well tolerated in the assay (Table 1).
™ ™
Note: This Qubit assay kit can be used with any Qubit Fluorometer.

Contents and storage

Cat. No. Q32851 Cat. No. Q32854
Component Concentration Storage[1]
(100 assays) (500 assays)

Qubit™ dsDNA HS Reagent 250 µL 1.25 mL 200X in DMSO 2°C to 8°C

(Component A)
Desiccate
Protect from light

Qubit™ dsDNA HS Buffer 50 mL 250 mL Not applicable ≤30°C

(Component B)

Qubit™ dsDNA HS Standard 1 mL 5 mL 0 ng/µL in TE buffer 2°C to 8°C[2]

#1 (Component C)

Qubit™ dsDNA HS Standard 1 mL 5 mL 10 ng/µL in TE buffer

#2 (Component D)
[1] When stored as directed, kits are stable for 6 months.
[2] For long-term storage, the dsDNA standards can be stored at ≤–20°C.

Required materials not supplied

• Nuclease-free pipettors and tips
™ ™
• Qubit Assay Tubes (500 tubes, Cat. No. Q32856) or Qubit Flex Assay Tube Strips (Cat. No. Q33252)

Critical assay parameters

Assay temperature
™
Qubit assays deliver optimal performance when all solutions are at room temperature; temperature fluctuations can influence the
accuracy of the assay.
™
To minimize temperature fluctuations, insert all assay tubes into the Qubit Fluorometer only for as much time as it takes for the
™
instrument to measure the fluorescence. Qubit Fluorometers can raise the temperature of the assay solution significantly, even over a
period of a few minutes. Do not hold the assay tubes in your hand before reading because this warms the solution and results in a
different reading.

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

Incubation time
™
To allow the Qubit assay to reach optimal fluorescence, incubate the tubes for the DNA and RNA assays for 2 minutes after mixing
the sample or standard with the working solution. After this incubation period, the fluorescence signal is stable for 3 hours at room
temperature when the samples and standards are protected from light.

Calibrate the Qubit™ Fluorometer

For each assay, you have the option to run a new calibration or use values from the previous calibration. To minimize variables that can
affect performance, performing a new calibration for every new assay run is strongly recommended. See Figure 1 for an example of the
calibration curve used to generate the quantification results.

Figure 1 The curve-fitting algorithm used to determine concentration in the Qubit™ dsDNA HS Assay.
™
The Qubit Fluorometer generates concentration data based on the relationship between the two standards used in the calibration. This plot
™
shows the line corresponding to the curve-fitting algorithm used in the calculation of concentration data for the Qubit dsDNA HS Assay. For
reference, the positions of the standards and a set of data points from an actual experiment are shown superimposed onto the line, demonstrating
that the curve-fitting algorithm gives accurate values for quantitation.

Assay selectivity
™
The Qubit dsDNA HS Assay is highly selective for double-stranded DNA (dsDNA) over RNA (Figure 2).

Figure 2 DNA selectivity and sensitivity of the Qubit™ dsDNA HS Assay.

™
Triplicate 10‑µL samples of λ DNA (Ο), E. coli rRNA (Δ), or a 1:1 mixture of DNA and RNA (∎) were assayed in the Qubit dsDNA HS Assay.
Fluorescence was measured at 485/530 nm and plotted versus the mass of nucleic acid for the DNA alone or RNA alone, or versus the mass
of the DNA component in the 1:1 mixture. The variation (CV) of replicate DNA determinations was ≤2%. The inset, a separate experiment with
octuplicate determinations, shows the extreme sensitivity of the assay for DNA. Background fluorescence has not been subtracted.

2 ™
Qubit dsDNA HS Assay Kit User Guide
Photostability of the Qubit™ reagents
™ ™
The Qubit reagents exhibit high photostability in the Qubit Fluorometer, showing <0.3% drop in fluorescence after 9 readings and
™
<2.5% drop in fluorescence after 40 readings. However, if the assay tube remains in the Qubit Fluorometer for multiple readings, a
temporary reduction in fluorescence will be observed as the solution increases in temperature (Figure 3). Note that the temperature inside
™
the Qubit Fluorometer may be as much as 3°C above room temperature after 1 hour. For this reason, if you want to perform multiple
readings of a single tube, remove the tube from the instrument and let it equilibrate to room temperature for 30 seconds before taking
another reading.

Figure 3 Plot of fluorescence vs. temperature for the Qubit™ dsDNA HS Assay.
™
The Qubit assays are designed to be performed at room temperature, as temperature fluctuations can influence the accuracy of the assay.

™
Qubit dsDNA HS Assay Kit User Guide 3
Effects of contaminating substances
™
A number of common contaminants have been tested with the Qubit dsDNA HS Assay, and most are well tolerated. For untested
contaminating substances, and, in general, for highest accuracy, the standards should be assayed under the same conditions as the
experimental samples. For example, if the experimental samples are in an unusual buffer and 10 µL of each sample is used, add 10 µL of
the unusual buffer (lacking dsDNA) to each standard.
Table 1 Effect of contaminants in the Qubit™ dsDNA HS Assay, tested over a range of 1–500 ng/mL.
DNA standards were assayed in the presence or absence of contaminants at the indicated final concentrations. Equivalent concentrations (approximate)
in 20‑µL or 10‑µL sample volumes are also listed. In all cases, results are given as OK, usually less than 10% perturbation.

Final concentration in the Concentration in 20‑µL Concentration in 10‑µL

Contaminant Result
assay sample sample

Sodium chloride 50 mM 500 mM 1M OK

Magnesium chloride 5 mM 50 mM 100 mM OK[1]

Sodium acetate 30 mM 300 mM 600 mM OK

Ammonium acetate 50 mM 500 mM 1M OK

Ethanol 1% 10% 20% OK

Phenol 0.1% 1% 2% OK[1]

Chloroform[2] 1% 10% 20% OK

SDS 0.01% 0.1% 0.2% OK

Triton™ X-100 0.01% 0.1% 0.2% OK[1]

dNTPs[3] 100 µM 1 mM 2 mM OK

BSA 10 mg/mL 100 mg/mL 200 mg/mL OK[1]

IgG 0.5 mg/mL 5 mg/mL 10 mg/mL OK

RNA 1X[4] 1X[4] 1X[4] OK

ssDNA 1X[4] 1X[4] 1X[4] OK

Oligos 1X[4] 1X[4] 1X[4] OK

[1] An acceptable result, but with some distortion of the standard curve; for best results, add the same amount of contaminant to the standard samples.
[2] Immiscible.
[3] A mixture of dATP, dCTP, dGTP, and dTTP.
[4] 1X indicates a concentration equal to the concentration of dsDNA.

Prepare samples and standards

™
This protocol provides instructions to prepare standards for calibrating the Qubit Fluorometer. If you plan to use the values of a previous
calibration, fewer tubes (step 1) and less working solution (step 3) will be needed (see “Calibrate the Qubit™ Fluorometer” on page 2).

IMPORTANT! For best results, ensure that all materials and reagents are at room temperature.
™ ™
1. Set up the required number of Qubit tubes for standards and samples. The Qubit dsDNA HS Assay requires 2 standards.
™
Note: Use only thin-wall, clear, 0.5-mL PCR tubes (Cat. No. Q32856) for the Qubit 4 Fluorometer and 8 × 200-μL tube strips (Cat.
™
No. Q33252) for the Qubit Flex Fluorometer.

2. Label the tube lids.

Note: Do not label the side of the tube as this could interfere with the sample read. Label the lid of each standard tube correctly.
™
Calibration of the Qubit Fluorometer requires the standards to be inserted into the instrument in the right order.
™ ™ ™
3. Prepare the Qubit working solution by diluting the Qubit dsDNA HS Reagent 1:200 in Qubit dsDNA HS Buffer. Use a clean plastic
™
tube each time you prepare the Qubit working solution.

IMPORTANT! Do not mix the working solution in a glass container.

4 ™
Qubit dsDNA HS Assay Kit User Guide
 ™
4. Add the Qubit working solution to each tube such that the final volume is 200 µL.

Standard assay tubes User sample assay tubes

Volume of working solution 190 µL 180–199 µL

Volume of standard 10 µL —

Volume of user sample — 1–20 µL

Total volume in each assay tube 200 µL 200 µL

™
Note: The final volume in each tube must be 200 µL. Each standard tube requires 190 µL of Qubit working solution, and each
™
sample tube requires anywhere from 180–199 µL. Prepare sufficient Qubit working solution to accommodate all standards and
samples.
For example, for 8 samples, prepare enough working solution for the samples and 2 standards: ∼200 µL per tube in 10 tubes yields
™ ™
2 mL of working solution (10 µL of Qubit reagent plus 1990 µL of Qubit buffer).
™
Qubit Fluorometers provide a reagent calculator, which quickly computes the necessary volume of working solution needed.
™
5. Add 10 μL of each Qubit standard to the appropriate tube.

6. Add 1–20 µL of each user sample to the appropriate tube.

Note: If you are adding 1–2 μL of sample, use a 2-μL pipette for best results.

7. Vigorously vortex for 3–5 seconds. Be careful not to create bubbles.

8. Allow all tubes to incubate at room temperature for 2 minutes, then proceed to read standards and samples (next section).

Read standards and samples

Follow the procedure appropriate for your instrument.

Read samples and standards with the Qubit™ 4 Fluorometer

™ ™
For a more complete overview on using the Qubit 4 Fluorometer, please refer to the Qubit 4 Fluorometer User Guide (Pub. No.
MAN0017209), available for download at thermofisher.com/qubit.

1. On the Home screen, touch dsDNA, then select dsDNA High Sensitivity as the assay type. Touch Read standards to proceed.
Note: If you have already performed a calibration for the selected assay, the instrument prompts you to choose between reading
new standards and running samples using the previous calibration. If you want to use the previous calibration, skip to step 4.
Otherwise, continue with step 2.

2. Insert the tube containing Standard #1 into the sample chamber, close the lid, then touch Read standard. When the reading is
complete (~3 seconds), remove Standard #1.

3. Insert the tube containing Standard #2 into the sample chamber, close the lid, then touch Read standard. When the reading is
complete, remove Standard #2.
Note: The instrument displays the results on the Read Standards screen. For information on interpreting the calibration results, refer
™
to the Qubit 4 Fluorometer User Guide (Pub. No. MAN0017209), available for download at thermofisher.com/qubit.

4. Touch Run samples.

5. On the assay screen, select the Sample volume and units.

a. Touch the + or – buttons on the wheel, or anywhere on the wheel itself, to select the sample volume added to the assay tube
(1–20 μL).
b. From the Unit dropdown menu, select the units for the output sample concentration.

6. Insert a sample tube into the sample chamber, close the lid, then touch Read tube. When the reading is complete (~3 seconds),
remove the sample tube. The top value (in large font) is the concentration of the original sample and the bottom value is the
™
dilution concentration. For information on interpreting the sample results, refer to the Qubit 4 Fluorometer User Guide (Pub. No.
MAN0017209).

7. Repeat step 6 until all samples have been read.

™
Qubit dsDNA HS Assay Kit User Guide 5
Read standards and samples with the Qubit™ Flex Fluorometer
™ ™
For a more complete overview on using the Qubit Flex Fluorometer, please refer to Qubit Flex Fluorometer User Guide (Pub. No.
MAN0018186), available for download at thermofisher.com/qubit.

1. On the Home screen, select dsDNA High Sensitivity (HS) as the assay type. Touch Read standards & run samples to proceed.
Note: If you have already performed a calibration for the selected assay, the instrument prompts you to choose between reading
new standards and running samples using the previous calibration. If you want to use the previous calibration, press Run samples
and skip to step 4. Otherwise, continue with step 2.

2. Insert the tube strip containing Standard #1 into the sample chamber, close the lid, then touch Run standards. When the reading is
complete (~3 seconds), remove Standard #1.

3. Insert the tube strip containing Standard #2 into the sample chamber, close the lid, then touch Run standards. When the reading is
complete, remove Standard #2.
Note: The instrument displays graphical results on the Standards complete screen. For information on interpreting the calibration
™
results, refer to the Qubit Flex Fluorometer User Guide (Pub. No. MAN0018186), available for download at thermofisher.com/
qubit.

4. Press Next from the Standards complete screen. When prompted, load the tube strips with your samples as shown in the Insert
samples screen. If you have fewer than 8 samples, touch to deselect the tube positions that do not contain a sample.

5. Select the units for the output sample concentration, then touch Next.

6. (Optional) Select More options to add the assay kit lot #, tags, or sample IDs. For information on using these options, refer to the
™
Qubit Flex Fluorometer User Guide.

7. In the Sample volume screen, enter the sample volume added to the assay tube (1–20 μL). Enter the volume directly in the Sample
volume text box, use the + and – buttons, or adjust the sample volume wheel to select the Sample volume added to the assay
tube.
Note: The sample volume used (1–20 μL) changes the assay accuracy range. A different sample volume or assay may be required if
the sample concentration is outside of what the assay can accurately quantify.

8. Insert a sample tube strip into the sample chamber, close the lid, then touch Run samples. When the reading is complete
(~3 seconds), remove the sample tube strip.
Standards and sample measurements are displayed on a graph with the results in a list below it.
Touch the graph icon to switch to the results list-only view. The values listed are the concentrations of the original samples. For
™
information on interpreting the sample results, refer to the Qubit Flex Fluorometer User Guide (Pub. No. MAN0018186).

9. Select Add samples and repeat step 8 to read more samples.

6 ™
Qubit dsDNA HS Assay Kit User Guide
Related products
Table 2 Assays

Product Quantitation range Quantity Cat. No.

Qubit™ Protein BR Assay Kit[1] 100 reactions A50668

0.1–20 mg
500 reactions A50669

Qubit™ Protein Assay Kit 100 reactions Q33211

12.5–5,000 μg
500 reactions Q33212

Qubit™ 1X dsDNA HS Assay Kit 100 reactions Q33230

0.1–120 ng
500 reactions Q33231

Qubit 1X dsDNA BR Assay Kit

™
100 reactions Q33265
4–4,000 ng
500 reactions Q33266

Qubit dsDNA HS Assay Kit

™
100 reactions Q32851
0.1–120 ng
500 reactions Q32854

Qubit™ dsDNA BR Assay Kit 100 reactions Q32850

4–2,000 ng
500 reactions Q32853

Qubit™ ssDNA Assay Kit 0.2–240 ng 100 reactions Q10212

Qubit™ RNA IQ Assay Kit 75 reactions Q33221

N/A
275 reactions Q33222

Qubit™ RNA HS Assay Kit 100 reactions Q32852

4–200 ng
500 reactions Q32855

Qubit RNA BR Assay Kit

™
100 reactions Q10210
10–1,200 ng
500 reactions Q10211

Qubit RNA XR Assay Kit

™
100 reactions Q33223
100–20,000 ng
500 reactions Q33224

Qubit™ microRNA Assay Kit 100 reactions Q32880

0.5–150 ng
500 reactions Q32881

Qubit™ 4 System Verification Assay 50 reactions Q33237

N/A
Kit

Qubit™ Flex System Verification 50 reactions Q33254

N/A
Assay Kit
[1] Qubit™ Protein BR Assay Kit is designed for use with Qubit™ 4 only.

Table 3 Instruments

Product Cat. No.

Qubit™ Flex Fluorometer Q33327

Qubit™ Flex Fluorometer NGS Starter Kit Q45893

Qubit™ Flex Fluorometer Quantitation Starter Kit Q45894

Qubit 4 Fluorometer
™
Q33238

Qubit™ 4 NGS Starter Kit Q33240

Qubit 4 Quantitation Starter Kit

™
Q33239

Qubit™ 4 RNA IQ Starter Kit Q33241

Qubit 4 Protein BR Starter Kit

™
A51292

™
Qubit dsDNA HS Assay Kit User Guide 7
Table 4 Consumables/Accessories

Product Quantity Cat. No.

Qubit Flex Assay Tube Strips

™
125 tube strips Q33252

Qubit™ Assay Tubes 500 tubes Q32856

Qubit 4 Fluorometer International Power Supply

™
1 each A36204
(replacement)

Qubit™ 4 USB Flash Drive 1 each Q46009

Limited product warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and
Conditions of Sale at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please
contact Life Technologies at www.thermofisher.com/support.

Life Technologies Corporation | 29851 Willow Creek Road | Eugene, Oregon 97402 USA
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,
PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0002326
Revision Date Description
C.0 8 March 2022 The format and content were updated.
B.0 5 March 2015 The content was updated.
A.0 February 2015 New document for the Qubit™ dsDNA HS Assay Kit.

Important Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all
applicable Limited Use Label Licenses.
©2022 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific, Inc. and its subsidiaries unless otherwise specified.

thermofisher.com/support | thermofisher.com/askaquestion
thermofisher.com
8 March 2022