MyQubit Assay Operation
Appearance of New Assays
New assays appear on the instrument in alphabetical order. On the Home screen, these appear from left to right and top to bottom. On the Upload screen, these appear from top to bottom. New assays that contain numbers will be arranged in numerical order, i.e., “Metals1” will appear before “Metals2.” In each case, new assays created using MyQubit firmware appear after the existing assays for DNA, RNA, and protein installed on the instrument.
The Dilution Calculator that is part of the existing assays for DNA, RNA, and protein quantification may not be suitable for use with new assays created with MyQubit. The Qubit® 2.0 Fluorometer reports values for the analyte concentration(s) in the actual assay tube inserted into the instrument. The Dilution Calculator enables the determination of analyte concentration in the starting sample, i.e., before dilution in the Qubit® assay tube. However, the existing Dilution Calculator has been optimized for aliquots of 1–20 μL in a final sample volume of 200 μL. Therefore, the Dilution Calculator will only remain accurate for new assays that adhere to this protocol.
Selection of Units
The ability to change the displayed units directly from the graphical user interface will be disabled for new assays created using MyQubit firmware. Units can be defined in the .qbt file.
All new assays requiring calibration that are created with MyQubit firmware will by default utilize the graphical display. The x-axis will be labeled at the origin with a value of 0 and on the far right with the value defined in the .qbt file as the second (high-end) calibration standard. Tickmarks will be visible at equally spaced intervals; however, these marks will not be accompanied by value labels. You may choose to Hide Graph at any time from the Results screen.
New assays that require calibration will be performed according to the same protocol as for the existing Qubit® assays. Standard #1 for every MyQubit assay is a sample blank. Standard 2 is a sample of a concentration to be determined by the user. After selecting your new assay(s) from the Home screen, follow the on-screen instructions to calibrate your assay.
*Note*: For the MyQubit assays available for download here, all parameters, including the concentration value of Standard 2, have been defined and are included in the appropriate .qbt files.
Please refer to the Qubit® 2.0 Fluorometer User Manual for a detailed description of instrument function and assay workflow.
For the purposes of MyQubit assay creation, we have defined accuracy as the average deviation of replicate samples from the expected concentration plus one standard deviation. In general, we have defined the optimal dynamic range for MyQubit assays as those concentrations that result in ≤15% accuracy given the above definition, or ≤10% average deviation alone.
You may wish to further optimize the algorithm constants to obtain greater accuracy, or to focus on a more specific range of analyte concentrations. For example, we have generally found that accuracy can be improved at one portion of the standard curve, i.e., lower concentrations; however, this simultaneously results in diminished accuracy at the opposite end of the standard curve, i.e., higher concentrations. We have defined the dynamic ranges for all of the Qubit® assays as those that result in the best accuracy possible across the broadest dynamic range.