Introduction

The software allows you to define which samples are present in each well of an experiment, as well as which targets, and which dyes are used to label each target. This sample and target data is then used to perform different analysis methods.

Samples

In the experiment we have created, select the Samples tab and you will see a blank display as shown below (note the screenshot is annotated). It may help to read through Editing Lists before reading this section as we will be adding items to lists.

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Samples Pane (1)

The Samples pane is where you will add samples to the experiment. These will be assigned a Color, a Name and an optional Note.

Adding Samples (2)

Add four samples to the experiment by clicking the “+” four times. These can be deleted or moved within the samples list, for information please refer to Editing Lists.

Setting Samples to Wells (3)

Samples can be Set to wells in two ways. Firstly, select Sample 1 then select the wells you wish to set to in the Wells as Table pane. Click on row A1 and drag through to A8, now click Set and you will see that they have now turned blue, each with Sample 1 as a name under the Sample column in the Wells as Table.

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Number Unassigned Wells (4)

Selecting Number unassigned wells will assign all unassigned wells with samples that have not yet been create numbered from sample 1-32, one sample per well. Click Number unassigned wells. 24 additional samples have been created from Sample 9 to Sample 32. This is because Sample 1 is already set for wells A1-A8, therefore the software created 24 additional ones and set them to the rest of the plate.

Clear Samples from Wells (5)

By selecting the Clear button, you will un-assign the sample you have selected from the well that was selected.

Now please repeat Setting Samples to Wells, setting Sample 2 to row B, Sample 3 to row C and Sample 4 to row D. Once complete you should be presented with a display similar to the one below.

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Targets

Targets are labelled with dyes that will emit fluorescence, sometimes at different wavelengths which help us differentiate between targets DNA. The Targets can be assigned to different Samples which contain the Targets involved. This process is controlled as followed:

Targets Pane (6)

The Targets pane is where you can add Targets to the experiment that can be assigned to specific wells.

Adding Targets (7)

Add a target to the experiment by clicking the “+” button.

Samples as Plate

Select the Samples as Plate tab to easily view what kind of target has been assigned to wells.

Assigning Targets to Wells as Standards (8)

Selecting wells to assign targets to is the same process as selecting wells when setting samples. Either highlight rows in Wells as Table or highlight wells in Samples as Plate. Once you have selected the wells you wish to assign, select the target you wish to assign and click Std. To view the different types of standards available please see the Standards section further on in the software manual.

Assigning Targets to Wells as Unknowns (9)

Now select A2 and with Target 1 selected click Unk. Notice that A2 contains a blue outer ring but does not have a symbol beside the well. This indicates it is now an unknown.

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Assigning Targets to Wells as Negatives (10)

Now select A3-A8 and click Neg. Note that A3 is now blue in Targets as Plate.

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Clearing Assigned Targets from Wells (11)

Targets can be cleared by selecting the target you wish to clear, selecting the wells and then clicking Clear. In the example below wells A4-A8 has been cleared of Target 1.

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Standards

When assigning a standard to a well you have several options in the type of standard you can assign. When defining known quantities of DNA you can create a dilution series or a constant concentration to all wells. Predefined controls or custom controls can also be assigned to your control wells depending on their amplification characteristics.

Known Quantity

A constant concentration of DNA can be assigned to one or more wells for a specific target. Multiple targets can be assigned to the same well per assignment, however multiple targets can not be used per assignment. To assign a known constant quantity select the target then the wells you wish to assign them to and select the Std. button. You will then be greeted with the following standards file dialog:

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By default the Constant field will already be selected. One thousand has been entered into the number field and 1E3 has appeared for the corresponding wells in the table below. To execute select OK. The selected well (A1 - A8) will now be assigned with 1E3 copies, indicated with a yellow + symbol next to the corresponding wells as shown below:

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Dilution Series

To apply a target to numerous wells as a dilution series select the target and wells you wish to assign and select Std. The order in which you choose the wells will be the order in which the dilution series will occur. In the example below, wells B1 - B8 were selected, meaning the dilution series will occur from B1 - B8. Once your wells are selected press the Std. button and you will see the standard file dialog as shown below:

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Initial quantity

This is the starting quantity of your dilution series standard. In the example above 1,000 has been entered which could correspond to 1,000 copies, 1,000 copies per uL or 1,000 pg.

Dilution Factor

This is the factor at which your standards will be diluted by. In the example above, the dilution factor is 10 which means each subsequent dilution will be diluted by a factor of 10.

Replicates

These are the amount of replicates that will be used per dilution. In the example above, 2 replicates per dilution were used.

To execute your dilution series press OK. In Samples as Plate your assigned wells will have a yellow + symbol next to the corresponding well which indicates a standard has been assigned to it. In Wells as Table the dilution quantity will be shown under the Target column, indicated with a image alt text symbol as shown below:

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Predefined Controls

A new feature to MyGo 3.0 is the introduction of predefined controls. A predefined control is assigned the same way a normal target is applied to a well. Select a well and a target you wish to assign and select Std. The following file dialog will appear:

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As you can see there are a list of predefined control for you to choose from. Their functions are as follows:

Positive Internal Control

This behaves like an “unknown”. Cq analyses will check that it amplifies, and display a warning otherwise.

Extraction Control

This behaves like an “unknown”. Cq analyses will check that it amplifies, and display a warning otherwise.

Internal Copy Number Control

This behaves like a normal “Standard”.

External Copy Number Control

This behaves like a normal “Standard”.

External Positive Control

This behaves like an “unknown”. Cq analyses will check that it amplifies, and display a warning otherwise.

External Negative Control

This behaves like a “Negative”. Cq analyses will check that it amplifies, and display a warning otherwise.

When a predefined control has been assigned the symbol in the Type column will reflect the specific predefined control assigned. IN the example below an external negative control has been assigned to well A1, with the abbreviation ENC.

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Custom Control

Custom controls give the user much more freedom over what the control looks for. They can choose whether the control is meant to amplify or not, if it is meant to amplify within a specific Cq range and even if the target melts within a specific Tm range. Custom colours and abbreviation can be assigned to your custom controls.

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Genotype Controls

Custom controls can be useful when geneotyping in HRM analysis. For example if you know the genotype of a specific sample then you can assign that sample with a custom control that will assign all samples that cluster in the same way to the same genotype. In the example below a Genotype Control 1 has has been added to the New Control field when assigning a custom control.

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To make this control active when genotyping you must select the Genotype Control selection as shown above. To apply this to your sample select Ok and your new custom control will be assigned to the experiment as shown below:

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Automatic HRM Analysis

When using a genotype control as described above the software will use this control to define an inferred genotype to a cluster in the auto HRM analysis module. Below the Genotype Control 1 was applied to well A1. Therefore, all other samples that clustered have been assigned with the same genotype as shown below:

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Manual HRM Analysis

This Genotype Control 1 can also be used in the manual HRM analysis as shown below:

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However, if a custom control is being used then this genotype can not be removed from the list of potential genotypes or overwritten by another genotype. If you wish to remove the custom control it must be removed in sample setup.

Multiple Targets

Multiple targets can be added to an experiment In the example below, a second target has been added and assigned to wells C1 - C8 as an unknown.

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Dye Conflict

If Target 1 and Target 2 were present in the same well, it would not be possible to distinguish them because they are both labelled with SYBR Green I. To demonstrate this, select wells B4 - B8, and with Target 1 selected in the targets pane click Unk. You will be presented with the following warning:

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Opening and Saving a Sample Layout (12)

Sample layouts can be opened and saved using the buttons below Wells as Table. Note that any sample layout that is opened will overwrite any sample layout that is already present in the Samples tab.

The sample layout can be stored in multiple formats, which can be selected in the save file dialog.

PCR Sample Data (.psd)

If saved as a psd. file, you will be able to load all information from the Sample tab but the sample layout file can not be edited externally.

Character Separated Variable (.csv)

All sample data (except sample and target colours) can be exported to, and imported from, .csv files.

RDML

The Real-time PCR Data Markup Language (RDML) is a structured and universal data standard for exchanging quantitative PCR (qPCR) data.