We have learned about different elements, now let's put them together for more complex workflows.
Mycoplasmas are some of the smallest known prokaryotic organisms, and common contaminants in mammalian cell cultures. Good laboratory practice and regulatory agencies require routine screening for mycoplasma. Different commercial kits are available for mycoplasma detection, including PCR- and luminescence-based assays.
In this tutorial, we will set up a luminescence-based assay for detection of mycoplasma in samples from cell culture.
In summary, samples from cell culture, positive, and negative controls are appended and aliquoted onto an assay plate. The mycoplasma detection reagent is added onto all wells followed by an incubation step. The plate is then read so that background luminescence is captured, before adding the mycoplasma substrate reagent, followed by another incubation and plate reading.
Get started
Create a workflow in the builder. To learn how, click here.
Rename the workflow Core elements tutorial 11.2. To learn how, click here.
Select the device on which you want to run your workflow. To learn how, click here.
Select a default plate type. To learn how, click here.
Note: Select two different plate types, a trough and a 96 well plate. This allows the platform to use a trough for liquids that require a higher volume, especially as the number of samples scales, and a 96’well plate for smaller volumes.
Define samples and reagents
We’ll start by defining the samples, reagents and controls using two Define Liquids and Plates elements. We use two elements to make the workflow more flexible: the number of samples is likely to vary, as is its input format (from a previous simulation or from an uploaded file, for eg), while reagents and controls are constant and their volume should automatically scale as the number of samples increases. For these reasons, it is better to use two instances of Define Liquids and Plates. For more information on how to use this element, click here.
In the workflow builder, define your samples and reagents using Define Liquids and Plates. In this example, samples are being defined using the table editor, and the reagents using the element parameters.
Aliquot samples and controls
Now we want to aliquot both the samples and the controls. However, we want to make sure that controls are aliquoted to all plates, so that if the number of samples increases, creating a new assay plate, the controls are also added to the new plate automatically. To do so, we will use two Aliquot elements and make use of the Replicate to all plates in set feature.
Add 4 elements to your canvas: 1x Select, 2x Aliquot and 1x Describe Layouts and wire them as such:
Use the liquid name to select the positive and negative controls. For more information on how to use Select, click here.
Specify the Aliquot Volume, Replicates, Plate Set Name and the Plate Type parameters in the Aliquot elements. We will aliquot a single replicate of the samples, but the controls will be aliquoted in duplicate.
Note: To apply a parameter value to all input liquids use the default identifier, as in the example above. To apply a parameter value to a specific liquid then provide the liquid name, or alternatively use a tag to apply a parameter value to a group of liquids. This behaviour is true for most parameters unless stated otherwise.
Because we want to make sure controls are automatically added to all plates as the number of samples increases, toggle Replicate To All Plates In Plate Set in the Aliquot Controls element.
Use Describe Layouts to define the plate format. For more information on how to use Describe Layouts, click here.
Mix reagent onto samples and controls, incubate and read background
Now that samples and controls have been aliquoted to the assay plate, we’ll mix the reagent onto all wells, incubate and read the background luminescence. First we need to append samples and controls, and again, we will use the Select element, as well as Mix Onto, Incubate and Run Plate Reader.
Add five elements to your canvas: 1x Append, 1x Select, 1x Mix Onto, 1x Incubate and 1x Run Plate Reader.
The Mycokit Reagent was defined in Define Reagents and then wired to a Select element, where the controls were selected. Therefore, we now need to use the Unselected Liquids parameter from the first Select element to the second one, and again select it based on liquid name.
Now let’s append the samples and the controls. Wire each of the Aliquots outputs to the Append element.
We want to add the reagent onto the plate with the samples and controls, so connect the Liquids output of the Append element to the Destination Liquids input of the Mix Onto element. Additionally, connect the Selected Liquids output of the Select element to the Source Liquids input of the Mix Onto element.
Click on the Mix Onto element to open the element parameter editor. In this case, we want each sample to receive the reagent, so we specify the addition mode to Mix In Order.
The new mixtures can inherit the names of the liquids already on the plates, in this case the samples, or you can specify a new name. In this case, we will not rename the mixtures so select the option Do Not Rename.
Now, we can define the volume of each reagent that should be added to the cell lines. In this case, the reagent should be added with the same volume, so specify a default value.
Importantly, you can specify a number of times, or replicates, you want the reagent to be added to the plate. For this protocol, this number is the number of liquids already on the plate. Considering that this is a workflow which could potentially be run with different numbers of samples, it is desirable to have the number of replicates scale automatically. To do so, instead of specifying a number, connect Output Liquid Count from the Append element to Replicates on the Mix Onto element. This way, the number of replicates will be the same as the number of samples, and automatically scale as the number of samples changes.
Now connect the output liquids from Mix Onto to the Incubate element and specify the incubation time.
Note: You may wish to use the Run Plate Reader element at this point to record the baseline for the assay. If you don’t have a plate reader in your lab integrated with Synthace’s Cloud Platform, you can substitute this element with a Prompt element.
After the first plate read, which measures the background luminescence, we want to add a second liquid, the substrate, on top of the existing liquids. Similarly to step 10, we now need to use the Unselected Liquids parameter from the second Select element and wire it to a new Select element, and again select it based on liquid name.
Now add a new Mix Onto element to the builder. Similarly to above, connect the Measured Liquids output of the Run Plate Reader element to the Destination Liquids input of the Mix Onto element. Additionally, connect the Selected Liquids output of the Select element to the Source Liquids input of the Mix Onto element.
Click on the Mix Onto element to open the element parameter editor. In this case, we want each sample to receive the substrate, so we specify the addition mode to Mix In Order.
The new mixtures can inherit the names of the liquids already on the plates, in this case the samples, or you can specify a new name. In this case, we will not rename the mixtures so select the option Do Not Rename.
Now, we can define the volume of each reagent that should be added to the cell lines. In this case, the substrate should be added with the same volume, so specify a default value.
As above, we want the substrate to be added to each liquid on the plate. To make sure the number of replicates automatically changes based on the number of samples inputted, instead of specifying a number, connect Output Liquid Count from the previous Mix Onto element to the Replicates parameter on the second Mix Onto element. This way, the number of replicates will be the same as the number of samples, and automatically scale as the number of samples changes.
Note: You may wish to use the Run Plate Reader element at this point to record the baseline for the assay. If you don’t have a plate reader in your lab integrated with Synthace’s Cloud Platform, you can substitute this element with a Prompt element.
Simulate the workflow
Check that the device that you selected can follow the instructions that you prepared. To learn how, click here.
Preview the execution
After you simulate the workflow, click View Simulation to open the simulation details.
Open the Preview tab, then click through the steps to check that the instructions that Synthace has generated are correct.
Advanced feature: Scale to a higher number of samples and change the number of replicates
Imagine you now want to make sure samples are added in triplicate. Which parameter(s) do you need to change?
Additionally what do you need to change if you want to run the workflow with 5 samples versus 200? How does that affect the input plate selection by the platform?