Thermo Fisher Scientific Qubit User Manual

USER GUIDE

Qubit™ 1X dsDNA HS Assay Kits

Catalog No. Q33230, Q33231

Pub. No. MAN0017455 Rev. C.0

Product information

Qubit™ 1X dsDNA HS (High Sensitivity) Assay Kits make DNA quantitation easy and accurate. The kits include a ready­to-use assay buffer and DNA standards. To perform the assay, simply dilute your sample (any volume from 1–20μL is
acceptable) into the 1X working solution provided, then read the concentration using a Qubit™ Fluorometer. The assay is
recommended for initial sample concentrations ranging from 10 pg/µL to 100 ng/µL providing a core detection range of

0.2 ng to 100 ng of DNA. Additionally, the assay is selective for doube-stranded DNA (dsDNA) over RNA (Figure 2, page

6) and tolerant of common contaminants such as salts, free nucleotides, solvents, detergents, or protein (Table 2, page

7) ). The assay is performed at room temperature, and the signal is stable for 3hours when the samples are protected
from light. In addition to the Qubit™ 1X dsDNA HS Assay Kits described here, we also offer other kits for assaying RNA,
protein, and dsDNA at a higher concentration range (Table 3, page 8) as well as the Qubit™ dsDNA HS Assay - Lambda
Standard (Cat. No. Q33233).

Note: To use the assay with the Qubit

from thermofisher.com/qubit.

Table 1. Contents and storage

Material

Qubit™ 1X dsDNA HS Working Solution
(ComponentA)

Qubit™ 1X dsDNA HS Standard #1
(ComponentB)

Qubit™ 1X dsDNA HS Standard #2
(ComponentC)

* When stored as directed, the kits are stable for at least 6months from the date of receipt.

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2 and 3 models, you will need to download and install the appropriate program file

Amount

Q33230

(100assays)

50mL 250mL 1X

1mL 5mL 0ng/μL in TE buffer

1mL 5mL 10ng/µL in TE buffer

Q33231

(500assays)

Concentration Storage*

• 2–8°C

• Protect from light

• 2–8°C

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

Materials required but not

provided • Nuclease-free pipettors and tips

Critical assay parameters

• Qubit™ Assay Tubes (Cat. No. Q32856)

• Qubit™ Flex Tube Strips (Cat. No. Q33252)

Assay temperature The Qubit

Incubation time To allow the Qubit

Photostability of Qubit™

reagents The Qubit

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1X dsDNA HS Assay delivers optimal performance when all solutions are
at room temperature (18–28˚C). 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; the 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.

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1X dsDNA HS Assay to reach optimal fluorescence, incubate the
tubes for 2minutes after mixing the sample or the standard with the working solution.
After this incubation period, the fluorescence signal is stable for 3hours at room
temperature when samples are protected from light.

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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. 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.

Qubit™ Fluorometer

calibration For each assay, you have the choice to run a new calibration or use the values from the

previous calibration. When you first use the instrument, perform a new calibration each
time. As you become familiar with the assays, the instrument, your pipetting accuracy,
and significant temperature fluctuations within your laboratory, you can decide how
comfortable you are using the calibration data stored from the last time the instrument
was calibrated. Additionally, remember that the fluorescence signal in the tubes
containing standards and samples is stable for no longer than 3hours. See Figure3
(page 6) for an example of the calibration curve used to generate the quantification
results.

Handling and disposal No data are currently available that address the mutagenicity or toxicity of the Qubit

1X dsDNA HS Reagent (the dye in Component A). This reagent is known to bind
nucleic acids. Treat the Qubit™ 1X dsDNA HS buffer with the same safety precautions
as all other potential mutagens and dispose of the dye in accordance with local
regulations.

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Qubit

1X dsDNA HS Assay Kits | 2

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Prepare standards and samples

This protocol assumes that you are preparing standards for calibrating the Qubit™
Fluorometer. If you plan to use the last calibration performed on the instrument (see
“Qubit™ Fluorometer calibration”, page 2), you need fewer tubes (step 1.1) and less
working solution (step 1.3).

1.1 Set up the required number of assay tubes (or tube strips) for standards and samples.
The Qubit™ 1X dsDNA HS Assay requires 2 standards.

Note: Use thin-wall, clear, 0.5-mL PCR tubes (Cat. No. Q32856) for the Qubit

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4 Fluorometer and 8 × 200 µL tube strips (Cat. No. Q33252) for the Qubit™ Flex
Fluorometer.

1.2 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.

1.3 Add 10µL of each Qubit™ standard to the appropriate tube.

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

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

1.5 Add the Qubit™ 1X dsDNA working solution to each tube such that the final volume is
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.
Ensure that you have sufficient Qubit™ working solution to accommodate all standards
and samples.

Note: To avoid any cross-contamination, we recommend that you remove the total

amount of working solution required for your samples and standards from the working
solution bottle and then add the required volume to the appropriate tubes instead of
pipetting directly from the bottle to each tube.

1.6 Mix each sample vigorously by vortexing for 3–5seconds.

1.7 Allow all tubes to incubate at room temperature for 2minutes, then proceed to “Read

standards and samples”. Follow the procedure appropriate for your instrument:

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