Core elements tutorial 3.5
Here we will cover the process of performing an in-place dilution of a defined set of liquids to a specific set of concentrations. In this example we will dilute 3 sets of PCR primers from a stock concentration down to a working concentration. For each primer set we will have a forward (fwd) and reverse (rev) primer. Two sets of primers will have a final concentration of 10 mM. The other will be 12.5 mM.
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 3.5. 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.
Define liquids
First, you must define the liquids required for the dilution process. These are i) the liquids you intend to dilute, ii) a diluent. For detailed instructions on defining liquids see here.
Open the Elements menu and search for the element called Define Liquids and Plates and click on the element tile to add an instance of this element to your canvas.
Click on the element to highlight it. This will open the parameters panel for this specific element instance.
In the parameters panel select Table Editor in the Define From parameter.
Click on Add Spreadsheet button to open the table editor.
You can define liquids by filling in the table. Here we have defined three liquids with Liquid Names, Concentrations and Concentration Units.
In order to dilute the samples in place we must specify the location and volume of the input liquids. To do this fill out the Volume, Volume Unit, Plate Name, Plate Type and Plate Location. Each liquid has been tagged with the Tag
ssDNA.
Once you have defined the liquids, click Done in the bottom right-hand corner.
For clarity re-name the Define Liquids and Plates element to
Define Primer Stocksin the Instance Name field.Repeat this process to define the liquid you intend to use as a diluent.
Rename the second instance of the Define Liquids and Plates to Define Diluent.
Dilute in place by concentration
Specify dilution behaviour - by concentration
Add the Dilute element to the workflow builder from the Elements menu.
Click on the Dilute element to display the element parameters for this instance.
Under Global Options specify the Dilution Location as In Place.
Set the Dilution Mode to Direct.
Set Describe Dilutions to By Concentration
Describe the liquids to dilute and specify parameter for dilution
We have now established how we would like to carry out the dilutions in Synthace. Now we will define which liquids we would like to dilute, what we will dilute them with and buy how much.
Connect the Liquids output from Define Primer Stocks to Liquids to Dilute.
Connect the Liquids output from Define Diluent to Diluent.
Specify the Diluent To Use Per Liquid parameter. In this case, we want to use the water we defined in Define Diluent for all our substrates.
Note: if you wish to override the liquid policy of the diluted liquids you can do so here.
4. Specify the Target Concentration. Set this to 12.5 mM for default. Now specify the target concentration for Primer_set_A_fwd and Primer_set_A_rev to 10 mM.
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 will see diluent added directly to your input samples to dilute them down to the specified concentration.
Dilute in place by factor
In addition to diluting in place By Concentration it is also possible to define the desired dilution By Factor and By Concentration Range. See below for instructions on altering the workflow to perform the dilutions by factor.
Set Describe Dilutions to By Factor.
Define the Dilution Factor.
Re-simulate the workflow and view the preview to see how the dilution behaviour has been changed when using Dilute - By Factor.
Check your work:
To see what your finished workflow to this tutorial should look like simply navigate to the Tutorials and search for Tutorial 3: Dilute.
Challenges
You want to perform a dilution in place but the well size of your input liquid is too small to allow this. How could you alter your dilution process to allow for this behaviour?