QuDye dsDNA HS Assay Kit manual

The kit is used for quantification of dsDNA with Qubit™ Fluorometer. QuDye dsDNA HS reagent selectively binds to double-stranded DNA, so nucleotides, single-stranded DNA, RNA, proteins, and other impurities do not impede the measurements. All reagents are optimized to perform the measurements with Qubit™ fluorometer, the measurement range of initial sample DNA concentrations is from 10 pg/µL to 100 ng/µL.

Kit components

Kit component Count
12102
100 assays
13102
100 assays (incl. tubes)
52102
500 assays
53102
500 assays (incl. tubes)
33010, QuDye dsDNA HS Reagent, 200x, 250 uL 1 1
B9650, Standard #1 / Quantitative standard, 0 ng/uL in TE buffer, 1 mL 1 1
B7650, dsDNA quantitative standard, 10 ng/uL in TE buffer, 1 mL 1 1
G2150, TE buffer, 20x, 5 mL 1 1
Polypropylene tube (0.5 mL thin-walled transparent) 100 500
63010, QuDye dsDNA HS Reagent, 200x, 1.25 mL 1 1
G9650, Standard #1 / Quantitative standard, 0 ng/uL in TE buffer, 5 mL 1 1
G7650, dsDNA quantitative standard, 10 ng/uL in TE buffer, 5 mL 1 1
N2150, TE buffer, 20x, 25 mL 1 1

Store at +4 °С. Warm to RT before use.

Shelf life 12 months.

! All measurements with QuDye dsDNA HS Assay Kit should be performed at room temperature (22–28 °С). Before starting, equilibrate all kit’s solutions to room temperature. Avoid warming the samples, as the sample temperature influences the measurement results; particularly do not hold the assay tubes in your hands just before fluorescence measurement with a Qubit™ fluorometer.

Protocol

  1. Prepare 1x TE buffer taking into account that 200 µL of 1x TE buffer will be required for each sample and for each of the two standards. In order to do that, dilute 20x TE concentrate 20-fold with deionized water.
  2. Prepare QuDye dsDNA HS dye working solution taking into account that 200 μL of dye working solution will be required for each sample and for each of the two standards. In order to do that, dilute QuDye dsDNA HS reagent concentrate 200‑fold with 1x TE buffer.
    For example, to measure 3 samples and 2 standards, prepare 200 μL x 5 = 1000 μL of 1x TE buffer and 1000 μL of dye working solution (mix 5 μL of QuDye dsDNA HS reagent concentrate and 995 μL of 1x TE buffer).
    ! It is recommended to use dye working solution within several hours after preparation. In case of postponed measurements protect prepared dye working solution from light.
    ! Use only plastic containers to prepare dye working solution, as QuDye dsDNA HS reagent can adsorb to glass surfaces, which results in decreasing of the dye concentration in samples and biases in the measurement results.
  3. Set up two 0.5 ml tubes (thin-walled and optical-transparent) for the standards and one tube for each sample. Label the tube lids. Do not label the side of the tube as this can interfere with the sample read.
  4. To each of two tubes for standards add 190 µL of QuDye dsDNA HS dye working solution and either 10 µL of dsDNA quantitative standard, 0 ng/µL (Standard #1) or dsDNA quantitative standard, 10 ng/µL (Standard #2). Vortex for 2–3 seconds and centrifuge briefly.
  5. To each tube for samples add 180–199 µL of QuDye dsDNA HS dye working solution and 20–1 µL of DNA sample, respectively (the total volume should be 200 µL). Vortex for 2–3 seconds and centrifuge briefly.
    The dilution of sample is optional and depends on its initial concentration. The initial sample concentration should be within the range of 10 pg/µL–100 ng/µL for measurement with a Qubit™ fluorometer. At the same time, avoid pipetting small volumes to dilute the initial sample in order to maintain accuracy and precision of your measurements.
  6. Incubate all tubes (containing standards and DNA samples) for 3–5 minutes at room temperature.
  7. Perform the fluorescence measurements.

Fluorescence measurement with a Qubit™ fluorometer

The next steps should be carried out according to the instruction of the Qubit™ fluorometer. Depending on the version of the fluorometer the menu items may differ from the specified below.

  1. On the Home screen of the Qubit™ fluorometer, choose «dsDNA HS (DNA High Sensitivity)» as the assay type. Press «Go»
  2. With each preparation of the dye working solution, calibrate the fluorometer. Select «Run new calibration» and press «Go».
  3. Insert the tube containing Standard #1 into the sample chamber, close the lid, then press «Go». When the reading is complete (~3 seconds), remove Standard #1. Insert the tube containing Standard #2 into the sample chamber, close the lid, then press «Go». When the reading is complete, remove Standard #2.
  4. Insert the tube containing user sample into the sample chamber, close the lid, then press «Go». On the screen you will see the QF value.
    Calculate the concentration of the sample by the formula: Concentration of the sample = QF value x 200/sample volume.

Related kits

QuDye dsDNA HS Assay Kit (optimized for Qubit®)

Ready kit optimised for Qubit® fluorometers featuring high sensitivity (HS) and a measurements range from 10 pg/uL to 100 ng/uL
Show pricing
Cat. # Quantity Price Lead time
12102 100 assays $75.00 in stock
13102 100 assays (incl. tubes) $90.00 in stock
52102 500 assays $270.00 in stock
53102 500 assays (incl. tubes) $335.00 in stock

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