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

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Answer

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 more accurate with the Qubit® fluorometer. The Qubit® Fluorometer-compatible kits are also less expensive per assay if you are processing fewer than 20 samples at a time.

Answer Id: E5015

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550941aaebd324d688c53937dbb56f78_FAQ

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

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Answer

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 mathematical algorithm in the Qubit® kit for generating the most accurate standard curve, so you just want to make a few dilutions of the highest standard curve in the kit so you can generate a curve from more points. This will give you better accuracy on another fluorometer.

Answer Id: E5016

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6d41993e0e849876c60460cbceeff265_FAQ

Can the Qubit® kit give an indication of sample quality?

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Yes, but the Qubit® kit does not give an A260/A280 ratio. The A260/A280 ratio is rather a vague measurement and there is no way to determine what the ratio 1.7 really means. However with the Quant-iT™ dyes, you will be able to determine a true ratio of DNA to RNA, find out how much DNA or RNA you have, and how much contamination nucleic acid you have. You can compare the Quant-iT™ dye reading to your A260 reading. If the A260 reading is higher than the concentration obtained from the Quant-iT™ assay (which often happens), then you have contamination.

Answer Id: E5017

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d6fb20fed90cbf2dca39d90601dd2d06_FAQ

Why are my UV absorbance readings higher than the Qubit® platform readings?

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Answer

UV absorbance readings will measure DNA, RNA, proteins, free nucleotides, excess salt, etc. Everything and anything that absorbs at 260 nm, in fact. Since the Qubit® Platform uses fluorescent dyes that preferentially bind DNA, RNA, or protein to generate a fluorescent signal, your concentration data will be more accurate than that obtained traditionally using absorbance-based methods. For additional information on the Qubit® and associated reagents, search for "Qubit" on the Life Technologies homepage.

Answer Id: E5018

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3b127a8d2c444385c85e942325ead35e_FAQ

Can the Qubit® Quantitation Platform do a good job of quantifying plasmids?

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Answer

Yes. If you are doing a typical plasmid miniprep with lots of DNA (over 50 ng/μL), you should use the Quant-iT™ DNA BR assay, as this will allow you to quantitate without making an extra dilution. If, on the other hand, you are doing “plasmid rescue” or some other method that gives you small amounts of DNA, then you should use the Quant-iT™ DNA HS assay.

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bd9a7d2336e310e4a99bbb5667e0cb74_FAQ

How does the accuracy and sensitivity of the Qubit® Quantitation Platform compare to a microplate reader?

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Answer

The accuracy and sensitivity of the Qubit® Quantitation Platform is 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.

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a8f155247019c011fec9ca680f270a05_FAQ

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

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Answer

Please see our suggestions below:

  • Make sure that you take your reading only after incubating for 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.

    Answer Id: E8147

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  • 4ac6f3b8e1abd2178ca052253f5ffd1e_FAQ

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

    Product FAQ

    Answer

    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 1 year 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.

    Answer Id: E8149

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    d9c2b607fc4bbe1322c2ec6367f79509_FAQ

    Why am I getting negative fluorescence values with my Qubit® Assays?

    Product FAQ

    Answer

    Negative fluorescence is a physical impossibility. It is an artifact from software autocorrecting for background signal. This means your reader is picking up and subtracting out background light at the cost of your data. Make sure to do a buffer-only control and assess the type of signal. You may need to switch to a different plate.

    Answer Id: E8153

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    d87d7b130c65b7825ebc5f64bc3a3bcb_FAQ

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

    Product FAQ

    Answer

    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.

    Answer Id: E8121

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    139b86a8693bd7f3933ab6dfcbb3a2bd_FAQ

    Can I make my own assay for the Qubit® Fluorometer?

    Product FAQ

    Answer

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

    Answer Id: E8122

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    4e11284ffea58b27ab8a2a150976ce81_FAQ

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

    Product FAQ

    Answer

  • Qubit® dsDNA HS Assay: 502 nm/532 nm
  • Qubit® dsDNA BR Assay: 510 nm/527 nm
  • Qubit® ssDNA Assay: 493 nm/518 nm
  • Qubit® RNA HS Assay: 644 nm/673 nm
  • Qubit® RNA BR Assay: 644 nm/673 nm
  • Qubit® microRNA Assay: 498 nm/518 nm
  • Qubit® Protein Assay: 470 nm/570 nm

    Answer Id: E8128

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  • e4c781354155c8e703ec0dc73643fc4c_FAQ

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

    Product FAQ

    Answer

    The Quant-iT™ PicoGreen® DNA assay is the oldest assay and the most general-purpose. It requires the most user preparation and calculation, but can be adapted for cuvettes or plates. The Quant-iT™ DNA Assays are designed for high throughput (>20 samples) and for use in 96-well plate readers with no further adaptation. The Qubit® assays are intended for low throughput (<20 samples), and are only used on the Qubit® Fluorometer. The Qubit® Fluorometer contains highly optimized algorithms that calculate the concentration of your sample for you. The performance of all of these assays is similar.

    Answer Id: E8129

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    b52feac836c4c47b68d20bd366f3cb59_FAQ

    Does DNA length have an effect on the dsDNA assays?

    Product FAQ

    Answer

    Yes, short fragments may not stain well and plasmid DNA results will vary depending on conformation.

    Answer Id: E8131

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    7eac725bc6ee28c9955cbc3744c5f1e1_FAQ

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

    Product FAQ

    Answer

    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.

    Answer Id: E8134

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