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View additional product information for Qubit™ dsDNA Quantification Assay Kits - FAQs (Q32853, Q32854, Q32851, Q32850)
24 product FAQs found
这里有一些建议：
1.在“查看标准品”或"查看校准"下查看标准品的原始荧光值（RFU）。确认样本的荧光值落在标准品的荧光值之间（或稍稍高于最高的标准品）。如果不是，那么样本已经超过了检测的精确范围。请参看产品目录中的各个检测试剂盒的置信区间。如果检测样本超过了置信区间，那么检测读数将是2位有效数字而不是3位。 要使样本进入准确测定范围，可稀释样本或使用更多或更少体积的样本（例如，如果样本读值较低的话，使用10 µL而非2 µL）。
2.检查温度因素。检测是温度敏感性的，荧光信号在较高温度时会减弱。样本之间或样本和标准品之间的温度波动可能引发问题。
确保缓冲液和保存在DMSO内的Qubit试剂处于室温。缓冲液和Qubit试剂应保存于室温，而不是冰箱内。保存于 4°C的缓冲液即使在室温放置2–3小时仍然不能达到室温。
确保你的样本和工作溶液不能过热（包括刚从离心机取出时）。保留在Qubit仪器内过久或多次读取的样本可能会升温。如果你想对一个试管多次读数，你应该将它从仪器内取出，放置30秒以平衡至室温，然后进行下一次读数。另外，在读数之前，不要将试管握在手中过长时间，因为这会加热样本，使读数变低。
3.确保正确制备Qubit工作溶液（使用试剂盒内的缓冲液1:200稀释）。确保正确制备标准品（10 µL标准品加入到190 µL工作溶液中）。确保试管中加入至少200µL液体（对于标准品和样本均是如此）。
4.确保你使用的试剂和标准品是6个月之内的，并且标准品保存方法正确。Qubit试剂存储溶液应尽可能避光保存。
5.确保在Qubit荧光计上选择的程序跟你所用的Qubit检测试剂盒是匹配的。
6.读取标准品和样本数值时，确保仪器的盖子完全关闭。
7.使用推荐的检测管（检测管不能阻挡仪器盖上盖子，并且检测管应当光学透明）。某些类型的检测管可能有较高的自发荧光而影响检测。
8.你在Qubit仪器中输入你所加入到工作溶液中的存储液的微升数了吗？如果输入了，那么Qubit荧光计获得此信息后给出的浓度是你的存储液的浓度。如果没有输入，那么你得到的浓度是检测试管（你放入Qubit荧光计的试管）内的浓度——不是你的存储液浓度。
9.如果你是将Qubit检测的结果和使用UV吸光值获得的浓度进行比较，并且基于UV吸光值的浓度显著较高，那么可能是因为核酸或蛋白污染。Qubit检测试剂对于DNA, RNA和蛋白的检测特异性比吸光值测定法高得多。
请看我们的下列建议：
•确保孵育2分钟后再读取读数（对于蛋白，孵育时间为15分钟）。
•如果你将试管留在Qubit 荧光计内并多次读数，那么读数将随着试管在仪器内的升温而降低。如果你需要读取多个读数，请将试管从仪器中取出，放置于试管架上，让它与室温平衡至少30秒，然后再重新读取读数。
•如果样本是避光保存的，那么你可以在混匀后3小时内读取读数。超过这一时间，读数将不准确。
•在读取间隔，将标准品和样本试管保存于黑暗避光处。
PicoGreen染料和其它基于荧光定量的试剂不建议用于对染料偶联的核酸进行定量。核酸上携带的染料基团会干扰定量试剂的结合或荧光产生。
大致在20-mer或更短范围内的链信号水平较低。对于大部分由短链组成的dsDNA样本，仍可使用试剂，但应使用与样本长度相当的dsDNA标准品。
Qubit荧光计拥有高度优化的算法，可以使用 Qubit assays 或 Quant-iT DNA assays为您计算样本的浓度。使用MyQubit固件， Quant-iT PicoGreen DNA Assay也适用于Qubit荧光计。所有这些检测试剂的性能表现是相似的。
Quant-iT PicoGreen DNA Assay是最成熟和最通用的检测试剂。它需要标准品DNA和缓冲液的稀释，但是可以使用比色皿，微孔板和NanoDrop 3300进行测定。
Quant-iT DNA Assay提供了一个现成的缓冲液和预稀释的标准品DNA，可以使用96孔的微孔板来分析大量样本(>20个样本)，而无需进一步的调整。< br / >
Qubit Assay适用于低通量（<20个样本）实验，并且仅能用Qubit荧光计读取数据。
精确的激发/发射波长信息室保密的，这里是激发/发射波长的大致信息：
- 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
是的，对于Qubit (1.0)荧光计之后的Qubit设备是可以的。点击此处（https://www.thermofisher.com/cn/zh/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/fluorometers/qubit/qubit-assays/myqubit.html）查看MyQubit检测说明。
通常来说，样本越干净越好。一些盐、蛋白、以及去垢剂不会影响检测，您可以查看特定的检测方案以了解哪些物质以及它们在哪些浓度下不会影响检测。
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 our suggestions below:
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.
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.
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Quantification Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Quantification Support Center.
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
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Quantification Support Center.
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.
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.
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Quantification Support Center.
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.
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.
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 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
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Quantification Support Center.
The working solution is stable for approximately 3 hrs.
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.
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.