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查看更多产品信息 Qubit™ 3 Fluorometer - FAQs (Q33216)
21 个常见问题解答
•样本超出了检测范围。请使用一个浓度更高的样本或使用较低的稀释度（例如，20 μL加入180 μL而非10 μL加入190 μL）。
•查看结果屏幕上的荧光值vs浓度图表，确认样本读值是落在标准品读值之间（参见使用手册）。
•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）。
•确保标准品试管和样本试管中加入了200 μL溶液。
•Qubit试剂和工作溶液避光保存。
•在Qubit 3.0荧光仪上选择和你所使用的Qubit检测试剂相匹配的程序并正确校准荧光计。标准品必须以正确的顺序使用。
•确保检测完全在室温下进行。
•样本超出检测范围。请使用较低浓度的样本。
•查看结果屏幕上的荧光值vs浓度图表，确认样本读值是落在标准品读值之间（参见使用手册）。
•读取标准品和样本读值时，确保仪器的盖子完全关闭。
•根据你所使用的Qubit检测试剂盒内的说明制备样本和标准品。
•确保检测完全在室温下进行。
使用薄壁透明的0.5 mL PCR管，例如Invitrogen的Qubit Assay管(http://www.thermofisher.com/order/catalog/product/Q32856) or Axygen PCR-05-C管子 (Fisher Scientific, Part Number 14-222-292)。其它类型的试管可能会有自发荧光，或为写字留的磨砂区域可能会干扰检测。
不需要。但是我们建议您在每次制备一个新的工作溶液时都使用新的标准品，以保证您在标准品中使用的工作溶液和您样本中使用的工作溶液是相同的。
Qubit 4.0荧光计有一个面积大、功能强的彩色触摸屏，可以无缝浏览整个工作流程。并可通过一个USB接口将数据导入USB存储器或直接导入电脑，实现高效的数据管理。另外，仪器有一个内置的试剂计算器，可以进行个性化设置为仅显示常用检测，添加新检测，包括使用MyQubit检测设计工具创建的用户自定义检测，并且可以设置显示语言为您选择的语言，包括英语，法语，西班牙语，意大利语，德语，简体中文以及日语。< / p >
< p >不可以。Qubit DNA和RNA试剂盒只能检测样本中DNA或RNA的含量。Qubit荧光计不能进行吸光度读数以提供A260/280比值或检测核酸样品中的蛋白质。这可以用NanoDrop仪器来完成。如果你的样品含有蛋白质或其他可以影响检测的污染物，应该进一步纯化。
如果你的样品中同时含有DNA和RNA，你可以使用(或同时使用)DNA和RNA Qubit试剂盒，并与用RNase或DNase处理过的样品进行比较，以获得各自DNA或RNA的准确结果。< / p >
UV吸光度读数测定的是所有在260nm有吸收值的分子，包括DNA，RNA，蛋白，游离核苷酸，芳香族氨基酸和其他碱基。Qubit荧光剂仅对你感兴趣的分子进行测定，所以其读数几乎总是小于A260读数。
NanoDrop设备利用紫外吸收检测法，不能区分DNA，RNA，游离核苷酸和其它污染物。Qubit荧光计利用专门设计的荧光检测技术，使用Molecular Probes染料对感兴趣的生物分子进行定量。这些荧光染料只有和特定的目标分子结合时才会发出信号，即使在低浓度也是如此。
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.
If you are not using the Ion Library Equalizer Kit for library normalization, one of the following can be used (but the library should be at 100 pM):
- Ion Library TaqMan Quantitation Kit
- Qubit 3.0 Fluorometer or Qubit 2.0 Fluorometer
- Qubit dsDNA HS Assay Kit
- Agilent 2100 Bioanalyzer Instrument
- Agilent High Sensitivity DNA Kit
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
We recommend two methods for gDNA quantification:
- Qubit 3.0 Fluorometer (Cat. No. Q33216) with Qubit dsDNA HS Assay Kit (Cat. Nos. Q32851, Q32854)
- qPCR using TaqMan RNase P Detection Reagents Kit (Cat. No. 4316831)
- The sample is out of range. Use a sample that is more concentrated or use a lower dilution (for example, 20 µL in 180 µL instead of 10 µL in 190 µL).
- View the Fluorescence vs. Concentration graph in the Results screen to confirm that the values for the samples fall between the values of the standards (see the manual).
- Ensure that you have prepared the Invitrogen 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 Qubit working solution).
- Ensure that the standard and sample tubes are filled to 200 µL.
- Protect the Invitrogen Qubit reagent and working solutions from light.
- Select the correct Invitrogen Qubit Fluorometer assay for the assay you are performing and calibrate the fluorometer correctly. Standards must be used in the correct order.
- Ensure that the assay is performed entirely at room temperature.
- The sample is out of range. Use a sample that is less concentrated.
- View the Fluorescence vs. Concentration graph in the Results screen to confirm that the values for the samples fall between the values of the standards (see the manual).
- Ensure that the lid is closed while reading standards and samples.
- Prepare samples and standards according to the instructions in the Qubit assay kit you are using.
- Ensure that the assay is performed entirely at room temperature.
For Qubit 4 and older instruments, use thin-walled, clear 0.5 mL PCR tubes such as Invitrogen Qubit Assay Tubes (http://www.thermofisher.com/order/catalog/product/Q32856). For Qubit Flex Fluorometers, use thin-walled 200 µL polypropylene tubes such as Invitrogen Qubit Flex Assay Tube Strips (http://www.thermofisher.com/order/catalog/product/Q33252). Other types of tubes can have auto-fluorescence or a frosted area for writing and may interfere with the assay.
No. But we do recommend using new standards every time you make a new working solution, so that the working solution used in your standards is the same as that used in your samples.
The Qubit 4.0 Fluorometer employs a large, robust color touch screen for seamless workflow navigation and exports data to a USB drive or directly to your computer via a USB cable for efficient data management. Also, the instrument has a built-in Reagent Calculator and can be personalized to show only the frequently used assays, to add new assays, including user-defined assays created with the MyQubit assay design tool, and to display in the language of your choice including English, French, Spanish, Italian, German, simplified Chinese, and Japanese.
No. 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.
The Qubit DNA and RNA kits only quantify the amount of either DNA or RNA in the sample. If your sample contains 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.
UV absorbance readings measure anything that absorbs at 260 nm, including DNA, RNA, protein, free nucleotides, aromatic amino acids and other bases. Quantification with the Qubit Fluorometer only measures the molecule you are interested in, resulting in a quantity that is almost always lower than the A260 reading.
The NanoDrop instrument uses UV absorbance, which cannot distinguish between DNA, RNA, free nucleotides, and other contaminants. The Qubit Fluorometer utilizes specifically designed fluorometric technology using Invitrogen dyes to quantitate biomolecules of interest. These fluorescent dyes emit signals only when bound to specific target molecules, even at low concentrations.