MyQubit Custom Assays on the Qubit® 3.0 Fluorometer
Easily create new Qubit® assaysThe MyQubit functionality allows you to create new assays for the Qubit® 3.0 Fluorometer in minutes, using parameters that can easily be uploaded to the instrument using a USB drive, without changing the existing assays. New assays can be optimized versions of existing fluorescence-based assays or can be completely novel.
Step 1: Collect data and define constants for MyQubit assays
Collecting the data
Read samples made using your assay reagents in Fluorometer mode on the Qubit® 3.0 instrument. Fluorometer mode allows you to manually select the excitation light source of your choice, blue LED or red LED, while reading fluorescence in both the green and far red emission channels. Raw fluorescence values are obtained to generate data for the development of your new assay.
Defining the constants
For your convenience, we have developed the “MyQubit EZ Guide” Excel® spreadsheet to process your Fluorometer mode data. The MyQubit EZ Guide allows you to enter Fluorometer mode readings collected from samples of known concentrations and then calculates constants that can be used to define the shape of the standard curve that will be used during the new assay’s calibration. We recommend that all users new to developing MyQubit assays use the MyQubit EZ Guide spreadsheet to generate the parameters for their assays.
Simply enter the fluorescence values obtained in Fluorometer mode on the Qubit® 3.0 Fluorometer, along with the corresponding concentrations of the analyte. Once replicate data has been entered into the spreadsheet, adjust the “N” and “K” parameters to optimize the curve fit algorithm. The goal is to adjust “N” and “K” to achieve the lowest possible “Quality score” (see example below).
Step 2: Define your assay
Creating the MyQubit file
To create your own MyQubit file go the MyQubit Assay Design Tool
In the MyQubit assay design tool, you enter the values for the various parameters of your custom assay and the tool will produce a .qbt file for upload to the instrument. (The "MyQubit File Parameters & Definitions" table below describes the different fields that comprise a MyQubit file and provides guidelines for defining them.)
Step 3: Upload your assay
MyQubit will guide you through the process of uploading new assays. New assays can be permanently uploaded without affecting existing applications.
Importing assays from a USB drive:
- To import one or more new assays, download the MyQubit assay file(s) (.qbt file) you wish to install to the root directory of a USB drive.
Do not download the.qbt file(s) to a folder in the USB drive.
- Insert the USB drive containing the new assay file(s) into the USB drive port of the Qubit® 3.0 Fluorometer.
- Go to the Settings screen and touch Import new assay. The instrument searches the USB drive for assay file(s) and the Import new assay screen displays the names of the assays found on the USB drive.
- Touch to select the assay(s) you wish to import. Touching the selected assay a second time deselects it. You can select more than one assay for import.
- Touch Next. The Import new assay screen displays the name of the assay being imported (or the first assay, if multiple assays were selected) and lists the destination folders where you can save them.
- Select the destination folder for the new assay and touch Save. The Verification screen opens (see Step 8). Each folder can hold up to 9 assays.
Alternatively, touch New folder to create a new destination folder (see Step 7).
- On the New folder screen, touch the New folder text field. A keyboard pops up. Using the keyboard, type in the name of the new destination folder. You can use up to 11 characters; spaces at the beginning or the end of the name are not recognized.
Touch Enter, and then touch Done to return to the Import new assay – Where screen. The newly created folder will appear in the list of folders.
Select the new destination folder and touch Save. The Verification screen opens (see Step 8).
- On the Verification screen, touch Done to complete the import process.
If importing only one assay, you will be returned to the home screen.
If importing multiple assays, you will be returned to the Import new assay screen and prompted to select the destination folder for the next assay in your import queue.
Importing assays from a computer:
When the Qubit® 3.0 Fluorometer is connected to a computer via a USB cable, MyQubit .qbt assay file(s) can be transferred directly from the computer to the instrument.
- Download the MyQubit assay file(s) (.qbt file) you wish to install.
- Connect the Qubit® 3.0 Fluorometer to the computer using a USB cable. The Qubit® 3.0 Fluorometer should be recognized as an external device by the computer.
- Copy and paste or click and drag the .qbt file(s) you wish to import to the Qubit® instrument window on your computer.
Note: The assays will not be visible on the Qubit® 3.0 Fluorometer until you perform the next step.
- Go to the Settings screen on the Qubit® 3.0 Fluorometer and touch Import new assay. The Import new assay screen displays the names of the assays found on the computer.
- Follow Steps 4–8 of the import procedure described in "Importing assays from a USB drive".
Note: The USB cable must remain connected to both the Qubit® 3.0 Fluorometer and the computer for the assay to be imported
Step 4: Perform your assay
Your new MyQubit assay is ready to use. Please refer to the Qubit® 3.0 Product Manual for a detailed description of instrument function and assay workflow.
|Assay Name||User defined, max 11 characters, alphabetical characters only||Indicates the name visible on the initial home screen, i.e., the "family" of assays. For example, "DNA" is an Assay Name on the factory-configured instrument.|
|Name Sub-text||User defined, max 17 characters, alphabetical characters only||Indicates any smaller sub-text under the assay name (or to the right of the assay name from the graphical display). For example, "High Sensitivity" is a Name Sub-text on the factory-configured instrument.|
|Calibration||YES or NO||Indicates whether you are creating an assay that requires calibration or not. "YES" indicates that the assay will require calibration. All assays created using MyQubit that require calibration will utilize two standards, the first of which is always a sample blank (analyte concentration of "0") and the second of which is a sample of a concentration to be determined by the user. "NO" indicates that the assay will not require calibration. In this case, the output will be a raw fluorescence value or values.|
|Units||User defined, text or "/" symbol only, max 7 characters||Only required when Calibration is "YES." Maximum length 7 characters.|
|Excitation||BLUE or RED||BLUE = 470 nm, filter 430–495 nm. RED = 635 nm, filter 600–645 nm.|
|Emission||GREEN, FAR RED, or BOTH||BOTH is only acceptable if Calibration is "NO." GREEN = 510–580 nm. FAR RED = 665–725 nm.|
|Std Signal Ratio Limit||User defined, numeric only, between 0 and 9999999999||Only needed when Calibration is "YES." This number specifies the amount, in raw fluorescence, that the signal for Standard #2 must exceed the signal for Standard #1 for your MyQubit assay. This is meant to help troubleshoot large oversights in calibration. If you are unsure of a reasonable threshold, or if this is not of concern to you, then enter a small value that you will be assured of attaining in most calibrations.|
|Low||User defined, numeric only, between 0.001 and 99999||Only needed when Calibration is "YES." Indicates the lowest sample concentration that the instrument will display a result for. Samples with concentrations below this value will be reported as "< Low" and will display the message "*Sample TOO LOW*".|
|High||User defined, numeric only, between 0 and 99999||Only needed when Calibration is "YES." Indicates the highest sample concentration that the instrument will display a result for. Samples with concentrations above this value will be reported as "> High" and will display the message "*Sample TOO HIGH*".|
|N||User defined, numeric only, between 0 and 9999999999||Only needed when Calibration is "YES." One of three algorithm constants that must be determined by the user. Indicates the linearity of the curve, with a value of 1.00 corresponding to completely linear.|
|K||User defined, a scaling factor, numeric only, between 0 and 9999999999||Only needed when Calibration is "YES." One of three algorithm constants that must be determined by the user. Indicates the portion of the curve to focus on.|
|S||User defined, numeric only, between 0 and 99999||Only needed when Calibration is "YES." One of three algorithm constants that must be determined by the user. Indicates the concentration value of Standard #2 used in assay calibration.|
|Green RFU Name||User defined, max 16 characters||Indicates the name given to the displayed RFU output for Green emission when Calibration is "NO." Only needed when Calibration is "NO" and Emission is "GREEN" or "BOTH."|
|Far Red RFU Name||User defined, max 16 characters||Indicates the name given to the displayed RFU output for Far Red emission when Calibration is "NO." Only needed when Calibration is "NO" and Emission is "FAR RED" or "BOTH."|
For Research Use Only. Not for use in diagnostic procedures.