Invitrogen™ Qubit™ dsDNA Quantification Assay Kits

Catalog No.	Quantity	Assay	Quantitation Range
Q32850	100 assays	dsDNA Quantitation, BR	4 to 2000 ng
Q32851	100 assays	dsDNA Quantitation, HS	0.1 to 120 ng
Q32854	500 assays	dsDNA Quantitation, HS	0.1 to 120 ng
Q32853	500 assays	dsDNA Quantitation, BR	4 to 2000 ng

Catalog No. Q32850 Supplier Invitrogen™ Supplier No. Q32850

Description

Includes

BR Assay Kit

Achieve accurate and precise quantification of double-stranded DNA with Qubit dsDNA HS and BR Assay Kits. These dsDNA quantification kits enable quick and selective detection of low and high abundance DNA samples, and can distinguish dsDNA from ssDNA, RNA, protein, and free nucleotides.

Achieve accurate and precise quantification of dsDNA with Qubit dsDNA BR (Broad Range) Assay Kits. These dsDNA quantification kits enable quick and selective detection of low and high abundance DNA samples, and can distinguish dsDNA from ssDNA, RNA, protein, and free nucleotides. Contaminants such as salts, solvents, or detergents are well-tolerated.

Qubit dsDNA HS and BR Assay Kits, designed for use with Qubit Fluorometers, are highly selective for double-stranded DNA (dsDNA) over single-stranded DNA (ssDNA), RNA, protein, and free nucleotides. All kits provide concentrated assay reagent, dilution buffer, and pre-diluted DNA standards. Simply dilute the reagent using the buffer provided, add your sample (any volume between 1 μL and 20 μL is acceptable), and read the concentration using a Qubit Fluorometer.

Qubit dsDNA HS Assay Kit

The Qubit dsDNA HS (High Sensitivity) Assay Kit, when used with the Qubit Fluorometer, provides an accurate and selective method for the quantitation of sensitive DNA samples. Depending on sample volume, the assay kit is designed to be accurate for initial DNA sample concentrations of 0.005 to 120 ng/μL, providing a detection range of 0.1-120 ng.

Qubit dsDNA BR Assay Kit

The Qubit dsDNA BR (Broad-Range) Assay Kit, when used with the Qubit Fluorometer, provides an accurate and selective method for the quantitation of DNA samples. Depending on sample volume, the assay kit is designed to be accurate for initial DNA sample concentrations of 0.2 to 2,000 ng/μL, providing a detection range of 4-2,000 ng.

Notes

• Qubit dsDNA HS and BR Assay kits can be used with any Qubit Fluorometer
• Use with thin-wall, clear, 0.5-mL PCR tubes (Cat. No. Q32856) for the Qubit 4 Fluorometer and 8 x 200-μL tube strips (Cat. No. Q33252) for the Qubit Flex Fluorometer

Specifications

• Assay: dsDNA Quantitation, BR
• Detection Method: Fluorescence
• Excitation/Emission: 510/527
• For Use With (Equipment): Qubit Fluorometer
• No. of Reactions: 100 Reactions
• Quantitation Range: 4 to 2000 ng
• Product Line: Qubit
• Quantity: 100 assays
• Shipping Condition: Room Temperature

Frequently Asked Questions (FAQs)

I'm seeing other kit-related problems besides the "Standards incorrect" message with my Qubit assay. What do you suggest I try?

Here are several suggestions:

1.View the raw fluorescence value (RFU) for the standards under “Check Standards” or “Check Calibration”. Confirm that the values for the samples fall between the values of the standards (or a little above the highest standard). If they do not, the sample is out of the accurate range of the assay. Refer to the confidence ranges for each assay in the product manuals. The readout in the assay will be to 2 significant figures instead of 3 if the assay sample is out of the high confidence range.
To bring the sample into the accurate range, dilute the sample or use more or less of it (for example, 10 µL instead of 2 µL if the sample reads low).

2.Check for temperature issues: The assay is temperature sensitive and the fluorescent signal can decrease at higher temperatures. Temperature fluctuations between samples, or between samples and standards, can cause problems. Make sure that the buffer and Qubit reagent in DMSO are at room temperature. The buffer and Qubit reagent should be stored at room temperature, not in the refrigerator. Even after 2-3 hours at room temperature, buffer previously stored at 4°C can remain below room temperature. Make sure your samples and working solution are not too warm (including those straight from a centrifuge). Samples kept in the Qubit instrument too long or read multiple times can warm up. If you want to perform multiple readings of a single tube, you should remove the tube from the instrument and let it equilibrate to room temperature for 30 seconds before taking another reading. Also, do not hold tubes in your hand for very long before reading them in the instrument, since this can warm the sample, resulting in a low reading.

3.Ensure that you have prepared the Qubit working solution correctly (1:200 dilution using the buffer provided in the kit). Ensure that you have prepared the standard tubes correctly (10 µL of each standard in 190 µL of the working solution). Ensure that the tubes are filled with at least 200 µL (both standards and samples).

4.Ensure that the reagents and standards you are using are less than 6 months old, and that the standards have been stored correctly. The Qubit reagent stock solution should be protected from light as much as possible.

5.Ensure that you have selected the correct assay on the Qubit Fluorometer for the Qubit assay you are performing.

6.Ensure that the lid is completely closed when reading standards and samples.

7.Use recommended tubes (both so the tube does not obstruct the lid, and for optical clarity). Some types of tubes can have high autofluorescence that will affect the assay.

8.Did you enter the number of microliters of stock you pipetted into the working solution into the Qubit instrument? If so, the reading after giving the Qubit Fluorometer this information is the concentration of your stock solution. If you did not, the reading you got is for the concentration in the assay tube (the tube you put into the Qubit Fluorometer) and not your stock solution.

9.If you are comparing Qubit assay results to concentration obtained by UV absorbance, and the concentration based on absorbance is significantly higher, it may be because of nucleic acid or protein contamination. The Qubit assays are much more specific for DNA, RNA, or protein than absorbance readings.

The value is decreasing over time when using the Qubit Fluorometer. What could be causing this?

Please see our suggestions below:

Make sure that you take your reading only after incubating for at least 2 minutes (15 minutes for protein).

If you leave the assay tube in the Qubit Fluorometer and take multiple readings, the readings will go down as the tube heats up inside the instrument. If you want to take multiple readings, remove the tube from the instrument, place it in a tube rack, and allow it to equilibrate to room temperature for at least 30 seconds before rereading the tube.

You may read the sample up to 3 hours after mixing if it is protected from light. After this time, the reading will not be accurate.

Keep standards and sample tubes in the dark and protected from light in between readings.

I'm trying to quantify some DNA labeled with a fluorophore. Will this work?

PicoGreen dye and other fluorescence-based quantification reagents are not recommended for quantifying dye-conjugated nucleic acids. The attached dye molecules can interfere with either binding and/or fluorescence output of the quantification reagents.

Does DNA length have an effect on the dsDNA assays?

Strands that are roughly in the 20-mer range or shorter show a lower level of signal. For dsDNA samples that are composed of mostly short strands, the reagent may still be used, but one should use a dsDNA standard that is of comparable length as the sample.

What is the difference between the Quant-iT PicoGreen DNA, Quant-iT DNA, and Qubit DNA assays?

The Qubit Fluorometer contains highly optimized algorithms that calculate the concentration of the sample using either the Qubit assays or the Quant-iT DNA assays. The Quant-iT PicoGreen DNA assay may be adapted to the Qubit Fluorometer using the MyQubit firmware. The performance of all of these assays is similar.

The Quant-iT PicoGreen DNA assay is the most established assay and the most general-purpose (http://tools.thermofisher.com/content/sfs/manuals/PicoGreen-dsDNA-protocol.pdf). It requires the dilution of the standard DNA and buffer but can be adapted for use with either cuvettes, microplates, or the NanoDrop 3300.

The Quant-iT DNA assays provide a ready-to-use buffer and pre-diluted standard DNA for analyzing a large number of samples (>20 samples) using a 96-well microplate with no further adaptation.

The Qubit assays (https://www.thermofisher.com/us/en/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/fluorometers/qubit/qubit-assays/myqubit.html) are intended for low throughput (<20 samples), and are only used on the Qubit Fluorometer.

What are the excitation/emission wavelengths for dyes in the Qubit Assays?

The exact excitation/emission wavelength information is proprietary. Here are the approximate excitation/emission wavelengths:

- Qubit dsDNA HS Assay: ~500 nm/ ~530 nm
- Qubit dsDNA BR Assay: ~510 nm/ ~530 nm
- Qubit ssDNA Assay: ~490 nm/ ~520 nm
- Qubit RNA HS Assay: ~640 nm/ ~670 nm
- Qubit RNA BR Assay: ~640 nm/ ~670 nm
- Qubit microRNA Assay: ~500 nm/ ~520 nm
- Qubit Protein Assay: ~470 nm/ ~570 nm

Can I make my own assay for the Qubit Fluorometer?

Yes, you can, for Qubit instruments developed after the original Qubit (1.0) Fluorometer. See MyQubit assay instructions here (http://www.thermofisher.com/us/en/home/life-science/laboratory-instruments/fluorometers/qubit/qubit-assays/myqubit.html.html).

I have a crude lysate. Will the Quant-iT and Qubit assays work?

Generally, the cleaner the sample the better. Some salts, proteins, and detergents are tolerated in the assays; see the specific assay protocol for which ones and at what concentrations.

How does the accuracy and sensitivity of the Qubit quantitation assays using the Qubit fluorometer compare to a microplate reader?

The accuracy and sensitivity of the Qubit quantitation assays are the same as that of a microplate reader. This was a requirement during product development. The detection limits for each Qubit kit can be found on the corresponding product manual, which can be found by searching our website by keyword or catalog number.

Can the Qubit kits give an indication of sample quality in nucleic acid samples?

No. The Qubit DNA and RNA kits only quantify the amount of either DNA or RNA in the sample. The Qubit fluorometer cannot take absorbance readings to provide a A260/A280 ratio or detect protein in nucleic acid samples. This can be done with the NanoDrop instrument. If your sample contains protein or other contaminants that can affect the assay, it should be further purified.

If your sample may contain both DNA and RNA, one may use either (or both) the DNA and RNA Qubit kits and compare with samples treated with either RNase or DNase to get an accurate determination of DNA or RNA, respectively.

Can I use the Quant-iT and Qubit Kits with other fluorometers?

All Quant-iT and Qubit kits are compatible with all fluorometers and microplate readers that have the appropriate light sources and filters. You won't have access to the algorithm in the Qubit fluorometer for generating the standard curve provided by the instrument, instead, you must make a few dilutions of the highest standard DNA or RNA (Standard #2) in the Qubit kits to generate a standard curve with multiple data points.

Can I use the original Quant-iT Kits with the Qubit Fluorometer?

No, we do not recommend this. Some of the dyes in the original Quant-iT kits (those NOT listed as “for use with the Qubit fluorometer”) are not compatible with the Qubit Fluorometer. In addition, the new Quant-iT kits (labeled as “for use with the Qubit Fluorometer”) have standards formulated to be compatible with the Qubit fluorometer internal algorithms for the respective assays. The Qubit Fluorometer-compatible kits are also less expensive per assay if you are processing fewer than 20 samples at a time.

The Qubit RNA High Sensitivity (HS), Broad Range (BR), and Extended Range (XR) Assay Kits (Cat. No. Q32855) and the Qubit dsDNA Quantification Assay Kits (Cat. No. Q32850) were both delivered at ambient temperature. Are they still usable?

Both kits are still usable if they were delivered at ambient temperature. However, once received, we recommend storing the individual components as indicated in the user guide(s).

What is the difference between The Qubit 1X dsDNA High Sensitivity (HS) and Broad Range (BR) Assay Kits (Cat. Nos. Q33230, Q33231, Q33265, Q33366) and Qubit dsDNA Quantification Assay Kits (Cat. Nos. Q32850, Q32851, Q32853, Q32854)?

The Qubit 1X dsDNA High Sensitivity (HS) and Broad Range (BR) Assay Kits (Cat. Nos. Q33230, Q33231, Q33265, Q33366) are newer and have a simplified workflow compared to the original Qubit dsDNA Quantification Assay Kits (Cat. Nos. Q32850, Q32851, Q32853, Q32854). The original Qubit kits contain separate dyes and buffer components that must be mixed together to make a working solution, in which the dye degrades after a few hours. With the newer Qubit 1X dsDNA Assay kits, the dye is premixed with a buffer that keeps the dye stable long-term and can be added directly to DNA samples.

You can find more information about the Qubit 1X dsDNA assay, by clicking on the Technical Note provided below:

Qubit 1X dsDNA assays: simplified workflow and improved performance

I am using the Qubit dsDNA BR Assay Kit. How long is the working solution stable for?

The working solution is stable for approximately 3 hrs.

Do you offer standard #2 that is included in the Qubit dsDNA BR Assay Kit as a stand-alone product?

Standard #2 of the Qubit dsDNA BR Assay Kit can be obtained separately as Qubit 1X dsDNA BR Assay Lambda Standards (Cat No. Q33263). Standard #1 is also included.

What is the shelf-life for the Qubit dsDNA BR Assay Kit?

When stored as directed (2-8 degrees C, protected from light), all our Qubit kits are stable for a minimum of six months (180 days) from date of receipt. Please refer to the user guide for further technical details.