The MyGo Pro software uses dye files to allow individual dye levels to be calculated based on the spectrum of light emitted from each well in the instrument. The instrument is supplied with a range of dyes already calibrated and available for use. The dye file generation analysis allows you to calibrate new dyes that are not included in the factory-calibrated list. The calibration is based on a PCR amplification using the relevant dye or labelled probe.
Performing Dye File Generation Run
Open the "*MyGo Pro Cq Precision.ppf"* run file from the MyGo Pro demo data folder and click on the Samples tab.
To create a dye file we recommend having the same sample in all wells as you can see below. All wells that will be used for dye calibration should be assigned the same sample, with a non-empty sample name. Furthermore, the name given to the Sample will be the name that appears in the software once it is opened. It is not necessary to assign a target to all the wells as the software will use the entire spectral data for each well to produce the dye calibration.
Select Analysis Type
Click the Analysis tab, Automatic and then add an FSO Auto Dye Calibration analysis from the Select Analysis Type window. If not already present learn how to do this in the Selecting Analysis Type section.
FSO Dye Calibration
Under the Analysis tab select the FSO Dye Cal. tab.
In the top left hand side of the screen is a window with two tabs labelled Dye Data and Amplification which contain settings for dye file generation.
Use Default Cycle Intervals
The Use default cycle intervals will be selected as default, as it chooses the optimum cycles to measure background and dye signals to produce the calibration.
The Use wells drop-down menu allows you to choose the wells that will be used to measure the dye spectrum to create a dye file. The software will automatically select all the wells for a specific sample.
The Amplification tab shows you the raw data from the PCR run. The total intensity uses average fluorescence data from all spectral channels. The software automatically chooses fluorescence data from an initial and a final set of cycles (highlighted) from each amplification curve.
The cycles shown in the Amplification graph are used to help obtain a spectral curve shown below:
The automatic dye file calibration tests if the dye spectra obtained from every well are sufficiently similar: if they are not, an error will be displayed. In the event that your experiment does not have sufficient quality we recommend repeating the experiment. Make sure that all wells contain the same reagents, volumes and have all been spun down correctly. Keep in mind that dye file quality will determine the quality of any subsequent experiments in which this dye file will be used, so it is important to ensure that the dye file data is really good data.
Exporting Calibration To File
This data can then be converted into a full spectrum dye calibration (.fsd) file by clicking the Export Calibration To File button as seen below.
Saving your Dye File
Choose a directory where you want to save your new dye file and select Save.
The user can specify a desired name for this dye file, as seen above as Dye File 1. However, the name that appears in the software will be the name that the user chooses to call the samples during Samples Setup.
Opening your Dye File
The generated MyGo Pro Dye File.fsd file can now be opened in the Dye Calibrations pane of the Run Settings tab. Note that the dye file is not automatically added to the run file used to create it, but can be added in the Run Settings tab. Once the dye file has been opened, it will be displayed in the list of Dye Calibrations, and can be assigned to targets in the run:
This dye file can now also be used in future runs.
Sending Dye Calibration to an Instrument
Dye Calibrations created in an experiment can be sent to a MyGo Pro instrument that is connected to your MyGo Pro software. Once an experiment has been executed the software will automatically search the instrument for potential dye calibrations that can be used in your analysis. If a dye calibration has been sent to an instrument then it will appear in the available Dye Calibrations shown above. Note that a dye file does not have to be sent to an instrument in order to use it in an experiment, as long as the dye file is available to open from a directory.
To send a Dye Calibration select Configuration in the top right hand side of the software window.
Once in Configuration select MyGo Pro where you will have multiple options available to you when configuring your MyGo Pro.
Manage Dye Calibration Files
Select the MyGo Pro you wish to configure, in this case 3A1F22CD08 and select Manage Dye Calibration Files:
Send A New Dye to Instrument
Now select Send a new dye to instrument and choose the dye you wish to send to your MyGo Pro from a directory*.* Select Open and the dye should appear in the list of Dye Calibrations as shown below:
Deleting Selected Dye On Instrument
Dyes can also be deleted from your MyGo Pro by selecting the Delete selected dye on instrument option. The dye will then be removed from the Dye Calibrations list. A deleted dye file can still be used if they are opened up in the experiment you wish to use them in. For more information on managing dye calibration files please refer to Configuring your MyGo Pro.
Dye File Availability
When creating many dye files for different experiments on multiple machines you can sometimes lose track of your dye files’ availability. This short section will explain the availability of the dye files you have created.
Dye Files Available in the Experiment
The dye files available in the experiment for analysis are the ones present in the Dye Calibration list under Run Settings. Before starting the experiment, there will be no dye calibrations, unless the experiment was opened from a template, in which case the calibrations from the template will be present. After starting the experiment, it will contain the calibrations it had before starting the run PLUS any calibrations stored on the instrument, PLUS calibrations stored in the software.
Where there are calibration duplicates the software will choose a calibration based on the following precedence (most preferred first):
Calibrations from the instrument.
Calibrations from the template.
Calibrations stored in the software.
Dye Files Available for Analysis
All dyes available in the experiment as described previously will be available for analysis. Dyes that are present on the instrument you are running from will be present in the dye calibration list and will be available for analysis. Dyes that have been seen an another instrument but not on the one you are using will not be present in the dye calibration list but will be shown in the target list. This dye will NOT be available for deconvolution until the dye file has been added to the experiment.