photo of Qubit® 2.0 Fluorometer with USB drive inserted  

Easily create new Qubit® assays

The MyQubit functionality allows you to create new assays for the Qubit® 2.0 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.

MyQubit functionality can only be implemented on Qubit® 2.0 Fluorometers running firmware v3.11 (or higher). For instruments requiring an upgrade, see firmware upgrade instructions.

How to create your own assay using MyQubit

Once you've developed your own fluorescence-based assay, you can create a Qubit® assay that will allow you to read your samples and directly translate the fluorescence values to concentration. Just follow the steps below.

Step 1: Enable Raw mode on your Qubit® 2.0 Fluorometer

screenshot from Qubit® 2.0 Fluorometer with Raw mode choice displayedWhen placed in Raw mode (called Fluorometer mode on the Qubit® 3.0 instrument), the Qubit® 2.0 Fluorometer can be used as a mini-fluorometer. In Raw mode, raw fluorescence values are obtained and can be used to generate data for the development of new assays or to validate the performance of existing assays. Qubit® 2.0 Fluorometers with serial numbers greater than 2286614942 are configured to operate in Raw mode. For Qubit® Fluorometers not configured for Raw mode, first download and save the Qubit® Raw mode file to your PC desktop (right-click, choose “Save As”) and then copy it to your USB drive. Ensure that this is the only .qbt file on the USB drive.

To upload the Raw mode configuration to the Qubit® 2.0 Fluorometer, turn the power off by unplugging the Qubit® 2.0 Fluorometer. While the instrument is unplugged, insert the USB drive into the instrument, and then plug the instrument back in. Your Qubit® 2.0 Fluorometer will detect the qubit_raw.qbt file present on the USB drive, and you will be prompted to upload the file. Once the upload is complete, you will be directed to a new home screen where Raw mode is now accessible.

Step 2: Collect data and define constants for MyQubit assays

Collecting the data

Raw mode allows you to manually select the excitation light source of your choice, i.e., blue LED, red LED, both, or none, while reading fluorescence in both the green and far red emission channels. Fluorescence values obtained in Raw mode can be used to generate data for the development of your new assay. For your convenience, we have developed the “MyQubit EZ Guide” Excel® spreadsheet to process your Raw mode data. We recommend that all users new to developing MyQubit assays use the MyQubit EZ Guide spreadsheet to generate the parameters for their assays.

Defining the constants

In addition to the straightforward parameters such as assay name and desired units output, several key constants must also be resolved prior to creating a new assay. These constants define the shape of the standard curve that will be used in the assay’s calibration. For your convenience, the “MyQubit EZ Guide” Excel® spreadsheet can be used to generate these constants based on Raw mode readings collected from samples of known concentrations. These constants can be entered into the MyQubit file so that the resulting standard curve is a good fit for the data you’ll be generating.

Simply enter the fluorescence values obtained in Fluorometer mode on the Qubit® 2.0 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).

         screenshot showing MyQubit EZ Guide spreadsheet completed with raw mode data

Step 3: Define your assay

Creating the MyQubit file

To create your own MyQubit file, download the MyQubit .qbt file template and save it to your computer.

Change the file extension from .qbt to .txt, and then use a simple text editor such as Notepad (Figure 1) to open it. Each MyQubit file can store information for up to four unique assays. The MyQubit file template* includes parameters for a single assay. To add additional assays, simply copy and paste the appropriate fields below the entries for assay “Name 1”. Additional assay name headers must be defined as “Name 2”, “Name 3”, and “Name 4”. Although all fields may not be populated for every assay, all field headers must be included for each assay in the MyQubit file in order for the file to upload properly. The MyQubit File Parameters & Definitions table below describes the different fields that comprise a MyQubit file and provides guidelines for defining them.

When you have entered all the parameters, close the file and change the extension back to .qbt. The file must be saved with the extension .qbt to be read by the Qubit® Flourometer. MyQubit files should first be created and saved external to the Qubit® 2.0 Fluorometer on a PC and then transferred to the USB drive provided with the instrument. The name of the file should not contain more than 20 characters from the standard ASCII library, excluding commas. Make sure there is only one .qbt file saved to the root directory of the USB drive. Having multiple .qbt files present on the USB drive will result in an error when uploading the file to the Qubit®2.0 Fluorometer.

*MyQubit file download instructions: 1) Click to download link; 2) Save to PC;  3) Open with a simple text editor such as Notepad, edit and re-save;  4) Upload to Qubit® with USB.
 

screenshot of text editor showing .qbt file parameter fields
Example of a .qbt file.

Step 4: Upload your assay

To upload your new assay, first turn the power off by unplugging the Qubit® 2.0 Fluorometer. While the instrument is unplugged, insert the USB drive into the instrument, and then plug the instrument back in. Your Qubit® 2.0 Fluorometer will detect the .qbt file present on the USB drive. If the file is correctly populated, you will be prompted to upload the assays contained in the .qbt file (Figure 2). Once the upload is complete, you will be directed to a new home screen where the Parent Name of your new MyQubit assay is visible.

three-panel instrument screenshot series showing steps to upload a new assay to the instrument

MyQubit will guide you through the process of uploading new assays. Up to 20 new assays can be permanently uploaded without affecting existing applications.

Step 5: Perform your assay

Your new MyQubit assay is ready to use. Please refer to the Qubit® 2.0 Fluorometer User Manual for a detailed description of instrument function and assay workflow.

MyQubit file parameters & definitions

Parameter Accepted values Description
Parent Name User defined, max 10 characters Indicates the name visible on the initial home screen, i.e., the “family” of assays.  For example, “DNA” is a Parent Name on the factory-configured instrument.
Name1 (and Name2, 3, 4) User defined, max 10 characters Indicates the individual assay name. For example, “dsDNA” is a Name on the factory-configured instrument.
Name Sub-text User defined, max 20 characters 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 only, max 10 characters Only required when Calibration is “YES.”  Maximum length 10 characters.
Excitation BLUE, RED, BOTH, or NONE BOTH and NONE are only acceptable if Calibration is “NO.”  Otherwise, this constitutes an error, and the .qbt file will not upload. 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.”  Otherwise, this constitutes an error and the .qbt file will not upload. GREEN = 510–580 nm. FAR RED = 665–725 nm.
Std Signal Ratio Limit User defined, numeric only 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 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 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 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 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 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 20 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 20 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.”

Addressing common errors associated with .qbt files

  • Make sure that there is only one .qbt file on the USB drive at a time.
  • Make sure that you have successfully upgraded your Qubit® 2.0 Fluorometer to v3.11 firmware.
  • Make sure that all necessary fields are completed in the .qbt file.
  • Make sure that all fields are included for each assay in the .qbt file, even if content is not required for your assay.
  • Create and edit the .qbt file externally using a PC, then transfer to a USB drive afterwards. Do not attempt to create or edit a .qbt file directly from the USB drive.
  • Make sure that the .qbt file is saved to the root directory of the USB drive.
  • Make sure that the field entries in your .qbt file adhere to the guidelines described in the MyQubit File Parameters & Definitions table.
  • Make sure that the name of your .qbt file does not exceed 20 characters and does not contain any commas.