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查看更多产品信息 Qubit® 2.0 Fluorometer - FAQs (Q32866)
12 个常见问题解答
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。
请参看下列可能性：
•如果你一直进行UV吸光值测定而你的读数小于预期，那么可能你的样本被其它有260nm吸光值的分子污染了。
•Qubit荧光计显示的最初数值是检测试管内的生物分子（DNA，RNA或蛋白）浓度。要获得样本中的生物分子浓度，请使用结果显示屏上的“计算存储液浓度” 或 ”计算样本浓度”功能。
•查看Qubit检测试剂盒产品手册，获取可能影响检测的污染物列表。
•读取标准品和样本数值时，确保仪器的盖子完全关闭。
•确保染料稀释液是新鲜配制（1 µL染料加入到199 µL缓冲液中）。
•确保正确标记试管。
•确保试剂盒未过期。
•确保染料保存于暗处。
•确保缓冲液和染料保存于室温。保存于4°C的缓冲液要在室温放置数小时使其平衡至室温。
•检查溶液中是否有气泡。溶液表面有气泡没关系，但是溶液内部或底部的气泡可能影响读值。
•为减少移液误差，稀释样本并使用较大体积。在操作高浓度或粘性溶液时这一点尤其重要。
最可能的原因是，样本被其它在260nm有吸光值的分子污染了。NanoDrop分光光度计读取这些污染物，但是 Qubit荧光计不会读取这些污染物。要确定样本的准确组成，可以对同一样本进行Qubit dsDNA Assay, Qubit RNA HS Assay, 和Qubit Protein Assay。查看Qubit检测试剂盒产品手册，获取可能影响检测的污染物列表。进一步稀释或纯化样本以降低污染物浓度。将工作溶液和加入其中的样本充分混匀。
是的，使用手册中有关于此项应用的说明。你将使用0 ng/μL lambda dsDNA HS标准品制备Standard #1。你将稀释一个10 ng/μL lambda dsDNA HS标准品得到Standard #2。然后你准备样本并将它们和上述的两点标准曲线进行比较。Quant-iT dsDNA BR试剂盒也可以用类似的方式使用。
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 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.