Get started
Read the introduction to this series of tutorials, then complete all of the previous tutorials in the series. For more information, click here.
Create a workflow in the builder. To learn how, click here.
Rename the workflow Core elements tutorial 5.2. To learn how, click here.
Select the device on which you want to run your workflow. To learn how, click here.
Prepare a luminol-based enzymatic assay
In this example, we will prepare four different mixtures to make up an luminol-based enzymatic assay and relevant controls, in replicates. In this case, because the ultimate goal is to measure the luminescence output of the reaction, it is important to add the different components in the right order to the mixture. To do so, we will use the element Make Mixtures using the addition mode Liquid Order.
The four mixtures are:
Enzyme negative control
Cells negative control
Elicitor negative control
Sample Reaction
The mixtures will contain:
PBS (make up to 200 ul)
Luminol (50 ul)
Horseradish peroxidase, HRP (15 ul, except for Enzyme Negative control)
Cells (100 ul, except for Cells negative control)
Elicitor (20 ul, except for Elicitor negative control)
In the workflow builder, define your mixture components using the Define Liquids and Plates element. For more information on how to use the Define Liquids and Plates, click here.
Add one element to your canvas: Make Mixtures.
Connect the Liquids parameter from the Define Liquids and Plates element to the Liquids To Make Mixtures input parameter of the Make Mixtures element.
Click on the Make Mixtures element to open the element parameter window. Here you can specify your mixtures. First, specify Parameters under Mixing Mode.
Now specify the composition of each mixture. To do so, give each mixture a name and add the names of the liquids defined in step 1.
Then, specify which liquid should be used as a diluent. The diluent will only be used if the mixture composition is such that you need to top up with some volume to make the desired final volume or concentration. This can be specified globally for all mixtures or individually for each one.
Next, we specify the target volume for each mixture. In this case this is our final reaction volume of 200 ul.
Note: The volume of PBS varies for each reaction, as some components are missing for some mixtures. Because PBS has been specified as the Diluent in step 6, the required volume of PBS will be automatically calculated as the difference between the target volume and the volume of all the components.
Select Liquid Order under Addition Mode and then specify the addition order of liquids. Use “default” to apply the order to all mixtures.
Finally, specify how the number of replicates and how the mixtures should be laid on a plate. Select Let Synthace Optimise from the dropdown menu for Layout and Replicate Grouping.
Define a Plate Set Name. This can be any alphanumeric name such as Mixtures.
Select a Plate Type from your plate library. Take care to ensure the plate type you select can accommodate the final volume for your mixtures.
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.
You can see that mixtures are prepared in replicates, and the individual liquids are added in the specified order:
PBS
Luminol
HRP
Cells
Elicitor
You can also easily inspect the composition of each mixture by selecting the corresponding well in the Setup view.
Check your work:
To see what your finished workflow to this tutorial should look like simply navigate to the Tutorials and search for Tutorial 5: Make Mixtures.
Challenges
Imagine now your protocol is described in terms of initial and final concentrations of each reagent. Can you modify the workflow above to achieve the following mixes:
PBS (make up to 200 ul)
Luminol (stock: 5 mM, final 50 uMl)
Horseradish peroxidase, HRP (source 10x, final 1x, except for Enzyme Negative control)
Cells (100 ul, except for Cells negative control)
Elicitor (stock: 1 ug/mL, final: 1 pg/mL, except for Elicitor negative control)