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View additional product information for Qubit® 2.0 Quantitation Starter Kit - FAQs (Q32871)
6 product FAQs found
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.
Please see the possibilities below:
- If you have been reading UV absorbance and your value is lower than expected, it is likely that your sample is contaminated with other molecules that absorb at 260 nm.
- The initial value that the Qubit Fluorometer shows is the concentration of the biomolecule (DNA, RNA, or protein) in the assay tube. To determine the concentration of the biomolecule in the SAMPLE, use the "Calculate Stock Concentration" or "Calculate Sample Concentration" feature on the results screen.
- Check the Qubit assay kit product manual for a list of contaminants that could interfere with the assay.
- Ensure that the lid is completely closed when reading standards and samples.
- Ensure that you have made a fresh dilution of the dye in buffer (1 µL dye to 199 µL buffer.)
- Ensure that you have labeled your tubes correctly.
- Ensure that the kit has not passed its expiration date.
- Ensure that the dye has been stored in the dark.
- Ensure that the buffer and dye are both stored at room temperature. It takes hours for a bottle of buffer at 4°C to warm to room temperature.
- Check for air bubbles in the solution. Bubbles on top are OK, but bubbles lower in the solution or at the bottom may affect the reading.
- To reduce pipetting error, dilute the sample and use a larger volume. This is especially important when pipetting concentrated or viscous solutions.
Most likely, the sample is contaminated with other molecules that absorb at 260 nm. The NanoDrop Spectrophotometer is reading these contaminants, but the Qubit Fluorometer is not. To determine the exact composition of the sample, perform a Qubit dsDNA Assay, Qubit RNA HS Assay, and Qubit Protein Assay on small aliquots of the same sample. Check the Qubit assay kit product manual for a list of contaminants that could interfere with the assay. Dilute or purify the sample further to reduce contaminant concentration. Mix the working solution and the sample aliquoted into it well.
Yes, the manual has directions for this application. You will use the 0 ng/µL lambda dsDNA HS standard to generate Standard #1. You will prepare a dilution of the 10 ng/µL lambda dsDNA HS standard to generate Standard #2. You then prepare the samples and compare them to this 2-point standard curve. The Quant-iT dsDNA BR Kit can be used in a similar manner.
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).
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.