Introduction

Fluorescent dyes or probes are used in qPCR to track and analyze data. Assays using the method of qPCR require fluorescence. To add a fluorescence, click on “Tests” and select “Fluorescence”. From there, click on “Add New Fluorescence”. For this tutorial we will make 3 fluorescent dyes.

Adding Fluorescence

• Step 1 - To add a fluorescence click on “Add New Fluorescence” and set the name as “Fluorescent 1”.
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• Step 2 - We will then repeat this process two more times for “Fluorescent 2” and “Fluorescent 3”.
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• Step 3 - Be aware that these names serve as an example. Realistically, actual fluorescent dyes such as FAM and TEX would be used.
• Step 4 - Your list of fluorescence should look similar to the following image.
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Introduction

Controls are a necessary component to every assay for analysis. Assays may have multiple controls that serve as a gradient. To add a control click on “Tests” and select “Controls”. From there, click on “Add New Control”. In this tutorial, we will first create a negative control (water) and then 7 other controls corresponding to different assays.

Adding Negative Control

• Step 1 - Let's first add our negative control that will only contain water. To do so, click on “Add New Control”, set the Name as “NC-H2O”, Lot Number as “LH2O”, and the Amount (µL) as 200.
• Step 2 - We will then repeat this process two more times for “Fluorescent 2” and “Fluorescent 3”.
• Step 3 - We will leave the Location empty and because controls tend to be the most time-sensitive components to PCR, we will set the Expiration Date 2 months ahead of today (your day).
• Step 4 - Keep in mind that every control must have a different lot number.
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Adding Assay Controls & Gradients

• Step 1 - Further in this in tutorial, we will be creating 5 assays and all will have control(s) assigned to them. All Expiration Dates for the following controls will be 1 day ahead of today (your day) for the sake of this demonstration. We will leave the Location empty.
• Step 2 - Let us first create “Control 1” with Lot Number as “L1” and the Amount (µL) as 200.
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• Step 3 - Second, “Control 2” with Lot Number as “L2” and the Amount (µL) as 200.
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• Step 4 - Third, “Control 3 G1” with Lot Number as “L3” and the Amount (µL) as 200.
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• Step 5 - Fourth, “Control 3 G2” with Lot Number as “L4” and the Amount (µL) as 200.
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• Step 6 - Fifth, “Control 3 G3” with Lot Number as “L5” and the Amount (µL) as 200.
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• Step 7 - Sixth, “Control 4 RNA” with Lot Number as “L6” and the Amount (µL) as 200.
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• Step 8 - Seventh, “Control 5 PCR” with Lot Number as “L7” and the Amount (µL) as 200.
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• Step 9 - Your list of controls should look similar to the following image.
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Introduction

Deciding what assays to run is a necessary step in PCR. Your samples may require a large variety of assays and organizing them can be a tedious task. PCRprep is designed specifically to handle this task with great efficiency. To create an assay click on “Tests” and select “Assays”. From the Assays page, click on “Create New Assay”. For this tutorial we will create a total of five assays. First we will make three assays for DNA in qPCR. Then one assay for RNA in qPCR and another one for DNA in PCR.

Creating Assay 1 DNA in qPCR

• Step 1 - For the first assay, click on “Create New Assay”, set the name as “Assay 1”, method as “qPCR”, and Type as “DNA”.
• Step 2 - We will set the Reaction Volume (µl) as 20 and the Sample Volume (µl) as 2.
• Step 3 - Since this assay method is for qPCR we will leave the the rest of the fields empty except for Replicates and Fluorescence.
• Step 4 - For now, we will leave Replicates as 1 and select “Fluorescent 1” under Fluorescence.
• Step 5 - We will then click on “Create Assay”.
• Step 6 - By doing so, we are able to update the assay with its corresponding controls and reagents.
• Step 7 - Let us add “Control 1” and “NC-H2O” as our controls.
• Step 8 - Then for reagents, we will select all reagents found in the following image except for the DNA template.
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• Step 9 - You first assay (Assay 1) should look similar to the following image.
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• Step 10 - Next, let's assign the final concentration of our reagents by clicking on the blue flask button near “Reagents”.
• Step 11 - Let us refer back to the image above to see what the final concentrations are.
• Step 12 - Let us input the final concentration for each applicable reagent. Note that PCRprep limits you to use the same concentration unit as the stock concentration. You will have to manually convert units accordingly.
• Step 13 - So for the Buffer, the final concentration is 1X, magnesium chloride is 1.5mM, dNTPs is 0.2mM, Forward Primer is 0.25µM, Reverse Primer is 0.25µM, and Polymerase is 1.25 Units.
• Step 14 - Your final concentrations should look similar to the following image.
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• Step 15 - You will also notice that you can change the order that the reagents will be displayed.
• Step 16 - You are also able to change the order of display of controls as well by going back to the assay edit page and clicking on the left-right arrow icon on top of your selected controls.
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Creating Assay 2 DNA in qPCR

• Step 1 - For the second assay, click on “Create New Assay”, set the name as “Assay 2”, method as “qPCR”, and Type as “DNA”.
• Step 2 - We will set the Reaction Volume (µl) as 20 and the Sample Volume (µl) as 2.
• Step 3 - Since this assay method is for qPCR we will leave the the rest of the fields empty except for Replicates and Fluorescence.
• Step 4 - For now, we will leave Replicates as 1 and select “Fluorescent 2” under Fluorescence.
• Step 5 - We will then click on “Create Assay”.
• Step 6 - By doing so, we are able to update the assay with its corresponding controls and reagents as shown below.
• Step 7 - Let us add “Control 2” and “NC-H2O” as our controls.
• Step 8 - Then for reagents, we will select all reagents found in the following image except for the DNA template.
• Step 9 - You second assay (Assay 2) should look similar to the following image.
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• Step 10 - We will then assign each reagent the exact same final concentrations according to the mastermix. Of course in a real scenario every assay will have different reagents and final concentrations. But for the sake of this demonstration we will simplify this process.

Creating Assay 3 DNA in qPCR

• Step 1 - For the third assay, click on “Create New Assay”, set the name as “Assay 3”, method as “qPCR”, and Type as “DNA”.
• Step 2 - We will set the Reaction Volume (µl) as 20 and the Sample Volume (µl) as 2.
• Step 3 - Since this assay method is for qPCR we will leave the the rest of the fields empty except for Replicates and Fluorescence.
• Step 4 - For now, we will leave Replicates as 1 and select “Fluorescent 3” under Fluorescence.
• Step 5 - We will then click on “Create Assay”.
• Step 6 - By doing so, we are able to update the assay with its corresponding controls and reagents as shown below.
• Step 7 - Assay 3 will have a gradient and so let us add “Control 3 G1, Control 3 G2, Control 3 G3”, and “NC-H2O” as our controls.
• Step 8 - Then for reagents, we will select all reagents found in the following image except for the DNA template.
• Step 9 - Your third assay (Assay 3) should look similar to the following image.
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• Step 10 - We will then assign each reagent the exact same final concentrations according to the mastermix.

Creating Assay 4 RNA in qPCR

• Step 1 - For the fourth assay, click on “Create New Assay”, set the name as “Assay 4”, method as “qPCR”, and Type as “RNA”.
• Step 2 - We will set the Reaction Volume (µl) as 20 and the Sample Volume (µl) as 2.
• Step 3 - Since this assay method is for qPCR we will leave the the rest of the fields empty except for Replicates and Fluorescence.
• Step 4 - For now, we will leave Replicates as 1 and select “Fluorescent 1” under Fluorescence.
• Step 5 - We will then click on “Create Assay”.
• Step 6 - By doing so, we are able to update the assay with its corresponding controls and reagents as shown below.
• Step 7 - We will select “Control 4 RNA”, and “NC-H2O” as our controls.
• Step 8 - Then for reagents, we will select all reagents found in the following image except for the DNA template.
• Step 9 - You fourth assay (Assay 4) should look similar to the following image.
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• Step 10 - We will then assign each reagent the exact same final concentrations according to the mastermix

Creating Assay 5 DNA in PCR

• Step 1 - For the fifth and last assay, click on “Create New Assay”, set the name as “Assay 5”, method as “qPCR”, and Type as “DNA”.
• Step 2 - We will set the Reaction Volume (µl) as 20 and the Sample Volume (µl) as 2.
• Step 3 - Since this assay method is for PCR, gels are required for further analysis. Knowing this, we must set the Ladder as “100bp Low Ladder” and Ladder Volume Per Gel (µl) as 2.
• Step 4 - If your ladder requires adding a dye for you to add then check “Dye Used In Ladder?”. Otherwise if your ladder is already pre-mixed with dye then leave the box unchecked. In this case, we will check the box.
• Step 5 - For Dye select “DNA Gel Loading Dye”, set Dye Volume Per Well (µl) as 1.5, and leave Replicates as 1. We will leave Fluorescence empty since this is only for qPCR assays.
• Step 6 - We will then click on “Create Assay”.
• Step 7 - By doing so, we are able to update the assay with its corresponding controls and reagents as shown below.
• Step 8 - We will select “Control 5 PCR”, and “NC-H2O” as our controls.
• Step 9 - Then for reagents, we will select the same reagents as the first assay we created.
• Step 10 - Your fifth assay (Assay 5) should look similar to the following image.
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• Step 11 - We will then assign each reagent the exact same final concentrations according to the mastermix
• Step 12 - Your list of assay should look similar to following image.
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Conclusion

Awesome! Once you have you created assays with controls and reagents, you are now ready to create your protocols. The Protocols section will instruct you how to create thermal cycler protocols, extraction protocols, and panels.