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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Read standards and samples

Qubit™ Flex Fluorometer

2.1 On the Home screen of the Qubit™ Flex Fluorometer, press the 1X dsDNA High Sensitivity (HS) assay icon. The “Read standards” screen is displayed. Press 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 2.4. Otherwise, continue with step 2.2.

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

2.3 Insert the tube strip containing Standard #2 into the sample chamber, close the lid, then press Read standards. When the reading is complete, remove Standard #2.

The instrument displays the graphical results on the Standards complete screen. For information on interpreting the calibration results, refer to the Qubit™ Flex Fluorometer

User Guide, available for download at thermofisher.com/qubit.

2.4 Press Next from the Standards complete screen. When prompted, load the tubes as

shown in the Insert samples screen. If you have fewer than 8 samples, press to deselect the tube positions that do not contain a sample. Select the units for the output sample concentration, then select Next.

Note: (Optional) Select More Options to add the assay kit lot number, Tags, or sample

IDs. For information on using these options, refer to the Qubit™ Flex Fluorometer User Guide.

2.5 In the Sample volume screen, enter the sample volume added to the assay tube (from 1-20 µL). Enter the volume directly in the sample volume text box, use the + or – 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.

2.6 Insert a sample tube strip into the sample chamber, close the lid, then press Run samples. When the reading is complete (~3seconds), remove the sample tube.

Standards and samples are displayed on a graph with the results in a list below. Select the graph icon only to view the results list. The value listed is the concentration of the original sample. For more information on interpreting the sample results, refer to the Qubit™ Flex Fluorometer User Guide.

2.7 Select Add samples to read more samples and repeat step 2.6.

Note: (Optional) Select Calculators to access the Molarity and Normalization calculators.

For information on the calculators, refer to the Qubit™ Flex Fluorometer User Guide.

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Read standards and samples

Figure 1. Workflow using Qubit™ Flex Fluorometer

Qubit™ 4 Fluorometer

3.1 On the Home screen of the Qubit™ 3 or the Qubit™ 4 Fluorometer, press DNA, then select 1X dsDNA HS as the assay type. The “Read standards” screen is displayed. Press 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 3.4. Otherwise, continue with step 3.2.

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

3.3 Insert the tube containing Standard #2 into the sample chamber, close the lid, then press Read standard. When the reading is complete, remove Standard #2.

The instrument displays the results on the Read standard screen. For information on interpreting the calibration results, refer to the Qubit™ Fluorometer User Guide, available for download at thermofisher.com/qubit.

3.4 Press Run samples.

3.5 On the assay screen, select the sample volume and units:

a. Press the + or – buttons on the wheel, or anywhere on the wheel itself, to select the

sample volume added to the assay tube (from 1–20μL).

b. From the unit dropdown menu, select the units for the output sample concentration.

3.6 Insert a sample tube into the sample chamber, close the lid, then press 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™ Fluorometer User Guide.

3.7 Repeat step 3.6 until all samples have been read.

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1X dsDNA HS Assay Kits | 5

Appendix

Selectivity of the

Qubit™1XdsDNA HS Assay

Figure 2. DNA selectivity and sensitivity of the Qubit™ 1X dsDNA HS Assay (Cat. Nos. Q33230, Q33231). Triplicate 10-μL samples of λ DNA (Cat. No. Q33233), E. coli rRNA, or a 1:1Mixture of DNA

and RNA were assayed with the Qubit™ dsDNA HS Assay. Fluorescence was measured and plotted versus the concentration of the RNA or DNA sample 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 is an expanded view of the low range of the assay showing the extreme sensitivity of the assay for DNA. Background fluorescence has not been subtracted.

How the Qubit™ Fluorometer

calculates concentration

Qubit™ assay kits compatible

with the Qubit™ Fluorometer

Figure 3. The curve-fitting algorithm used to determine concentration in the Qubit™ 1X 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 (a modified Hill plot) used in the calculation of concentration data for the Qubit™ 1X 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. The application note "Comparison of Quant-iT™ and Qubit™ quantitation assays for accuracy and precision" (available at thermofisher.com/qubit) describes in greater detail how the optimized Qubit™ algorithm provides improved accuracy and precision at the low end of the concentration range (<10ng/mL) over traditional 8-point standard curve and linear regression.

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1X dsDNA HS Assay Kits | 6

Contaminants tolerated by the

Qubit™ 1X dsDNA HS Assay

Note: While the contaminant tolerances of Qubit™ 1X dsDNA HS assay and the Qubit™ dsDNA HS assay are largely similar, they are not identical.

Table 2. Effect of contaminants in the Qubit™ 1X dsDNA HS Assay*

Contaminant

Final concentration

in the assay

Concentration in

10-μL sample

Concentration in

1-μL sample

Result

Sodium chloride 50mM 1M 10M OK

Magnesium chloride 1mM 20mM 200mM OK

Sodium acetate 30mM 600mM 6 M OK

Ammonium acetate 25mM 500mM NA OK

Sodium azide 1mM 20mM 200mM OK

Ethanol 1% 20 % NA** OK

Phenol 0.1% 2% 20% OK

Chloroform

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1% 20 % NA** OK

SDS 0.002% 0.04% 0.4% OK

BSA 1X

IgG 1X

ssDNA 1X

RNA 1X

dNTPs

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* DNA standards were assayed in the presence or absence of contaminants at the indicated final concentrations. Equivalent concentrations (approximate) in 10μL or 1μL sample volumes are also listed. In all cases, results are given as OK, usually less than 10% perturbation. For best results, add the same amount of contaminant to the standard samples.

** User sample would require greater than 100% of listed contaminant. † Immiscible. ‡ 1X indicates a concentration equal to the concentration of dsDNA.

§ A mixture of dATP, dCTP, dGTP, and dTTP. NA: Not available.

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Qubit assay kits compatible

with the Qubit™ Fluorometer

A number of fluroescence-based quantification kits are available for use with the Qubit™ Fluorometer. Use Table 3 to choose a kit based on the target molecule being measured and the number of assays you require.

Table 3. Qubit™ assay kits for use with the Qubit™ Fluorometer

Product Cat. No.

Qubit™ 1X dsDNA BR Assay Kit

Qubit™ dsDNA HS Assay Kit

Qubit™ ssDNA Assay Kit

Qubit™ RNA HS Assay Kit

Qubit™ RNA BR Assay Kit

No, of

assays*

Target Notes

Q33265 100

dsDNA

Q33266 500

Q32851 Q33230

100

dsDNA

Q32854 Q33231

500

Q10212 100 ssDNA

Q32852 100

RNA

Q32855 500

Q10210 100

RNA

Q10211 500

• Core range (high conﬁdence): 0.01µg/mL to 5µg/mL

• Extended range (moderate conﬁdence): 5µg/mL to 10µg/mL

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• Useful for quantitation of genomic and miniprep DNA samples

• Accurate in the presence of RNA, salts, solvents, proteins, and free nucleotides

• Core range (high conﬁdence): 1ng/mL to 500ng/mL

• Extended ranges (moderate conﬁdence): 0.5ng/mL to 1ng/mL and 500ng/mL to 600ng/mL

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• Useful for quantitation of PCR products, viral DNA, and samples for subcloning

• Accurate in the presence of RNA, salts, solvents, proteins, and free nucleotides

• Core range (high conﬁdence): 5ng/mL to 1000ng/mL

• Extended ranges (moderate conﬁdence): 1 ng/mL to 5ng/mL and 1000ng/mL to 1200ng/mL

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• Useful for quantitation of oligos, primers, denatured DNA, PCR products

• Accurate in the presence of salts, urea, solvents, proteins, ATP, and agarose

• Core range (high conﬁdence): 25ng/mL to 500ng/mL

• Extended ranges (moderate conﬁdence): 20ng/mL to 25ng/mL and 500 ng/mL to 1000ng/mL

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• Useful for quantitation of samples for microarray, RT-PCR, and Northern blot procedures

• Accurate in the presence of DNA, salts, solvents, proteins, and free nucleotides

• Core range (high conﬁdence): 0.1µg/mL to 5µg/mL

• Extended ranges (moderate conﬁdence): 0.05µg/mL to 0.1µg/mL and 5µg/mL to 6µg/mL

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• Useful for quantitation of samples for microarray, RT-PCR, and Northern blot procedures

• Accurate in the presence of DNA, salts, solvents, proteins, and free nucleotides

• Core range (high conﬁdence): 5ng/mL to 500ng/mL

• Extended ranges (moderate conﬁdence): 2.5ng/mL to 5ng/mL and 500ng/mL to 750ng/mL

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• Useful for quantiﬁcation of samples for qRT-PCR and sequencing applications

• Accurate in the presence of rRNA, large mRNA (>1000bp), salts, solvents, proteins, and free nucleotides

Qubit™ microRNA Assay Kit

Q32880 100

RNA

Q32881 500

• Core range (high conﬁdence): 1.25µg/mL to 25µg/mL

Qubit™ Protein Assay Kit

Q33211 100

Protein

Q33212 500

• Extended ranges (moderate conﬁdence): 1µg/mL to 1.25µg/mL and 25 µg/mL to 26µg/mL

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• Little protein-to-protein difference in signal

• Accurate in the presence of DTT, β-mercaptoethanol, amino acids, and DNA

• Signal is stable for 3hours

• Although small in size, the tertiary structure of 5s and tRNA will bind the large RNA dye

• Accurate in the presence of salts, protein, solvents and RNA stabilization reagents

• Signal is stable for 1hour

• The Qubit™ RNA IQ Assay will only work on the Qubit™ 4 or Flex Fluorometer. The

Qubit™ RNA IQ Assay Kit

Q33221 75

RNA

integrity

and quality

Q33222 275

assay is not designed for use with Qubit™, Qubit™ 2, or Qubit™ 3 Fluorometers

*Based on an assay volume of 200µL.

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Concentration ranges refer to the concentration of sample after dilution in the assay tube.

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1X dsDNA HS Assay Kits | 8

Ordering information

Cat.No. Product name Unit size

Q33230 Qubit™ 1X dsDNA HS Assay Kit, 100 assays ............................................................. 1kit

Q33231 Qubit™ 1X dsDNA HS Assay Kit, 500 assays ............................................................. 1kit

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