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Mix liquids based on sample metadata

Core elements tutorial 12.2

Updated over 8 months ago

In this tutorial, you will learn how to use sample Tags and metadata values to control the mixing of liquids. This type of liquid handling behavior is a powerful tool as it allows you to specify a decision point in a workflow and use information about a sample to drive how that, or group of samples is processed in the workflow. In-workflow decision-making also enables scalability.

We will explore the principles discussed in the overview using the example of heat shock transformation of bacteria with plasmid DNA and subsequent antibiotic selection. In this process, the correct antibiotic must be added to the correct bacterial culture. To ensure the workflow always adds the correct antibiotic we will build this logic into the workflow itself, thus removing the name for the user to specify this information and removing the need to refactor the workflow for each different antibiotic-plasmid combination.

  1. Get started

  2. Build a basic transformation workflow

    1. Reconfigure the existing workflow

    2. Define metadata and bacterial strains

    3. Heat Shock Process

    4. Reducing sample waste from residual volume

    5. Mixing liquids based on sample metadata

  3. Simulate the workflow

  4. Preview the execution

  5. Check your work

  6. Building flexible and scalable workflows

  7. Challenges

Get started

  1. Read the introduction to this series of tutorials, then complete all of the previous tutorials in the series. For more information, click here.

  2. Create a copy of the workflow that you created in Core elements tutorial 12.1. To learn how, click here.

  3. Rename the copy Core elements tutorial 12.2. To learn how, click here.

Build a basic transformation workflow

Heat shock transformation consists of 4 main steps:

  1. DNA addition and incubation

  2. Heat shock

  3. Recovery

  4. Selection

Reconfigure the existing workflow

  1. Delete the Aliquot All Liquids element from the workflow we built in Core elements tutorial 12.1

Define metadata and bacterial strains

Here we will describe the bacterial strains we wish to transform, expand the liquid definition of the plasmid DNA to include the correct metadata, and construct a workflow to execute the transformation protocol.

  1. Define the samples you wish to add to previously prepared samples in transformation_plate. Add a Define Liquids and Plates element and rename it to Define Chassis Cells. For this example, we have defined 2 wells in a plate containing our chassis cell which will be transformed with the plasmid DNA.

  2. Aliquot 8 ul of the cells to a new plate. Ensure you Keep Replicates Together and define the plate contents to ensure all the chassis cells are aliquoted By Column.

  3. Add a Mix Onto element to the workflow builder and name the element Mix DNA onto Cells.

  4. Connect the liquids defined in Define Chassis Cells to Liquids in Place.

  5. Connect the DNA samples from Append to the input Liquids to Add.

Heat Shock Process

  1. In order to describe the heat shock, we will use 3 Incubate elements.

  2. Add 3x Incubate elements to the workflow builder and define 3 incubation stages, a 5-minute incubation at 4 °C, 1 minute at 42 °C and a second 5 minute at 4 °C. Name each Incubate element to something descriptive.

Reducing sample waste from residual volume

Now that you have performed the heat shock of the cell-DNA mixture we want to add our cells to the recovery media. However, due to the residual volume of the plate, which this mixture is contained, we are not able to remove the entire well contents. To avoid wasting so much of our sample we will add recovery media onto the chassis-DNA mix, and then transfer this suspension, meaning a small proportion of the heat-shocked cells are wasted.

  1. Define a Recovery Media and aliquot on the chassis-DNA mix. In this example, we have defined the Recovery Media - SOC. Name this Define Liquids and Plates element Define Recovery Media.

  2. Mix 190 ul of this (or as much as your plate will allow) onto your heat-shocked cells using Mix Onto. First, select Mix All to All as the Mix Mode, define the Transfer Volume and connect the Liquids from Define Recovery Media. Rename this element to Add Recovery Media to Cells.

  3. Into another plate, Aliquot 300 ul of the recovery media, rename the element to Aliquot to Recovery Plate. This plate will be used to incubate and recover the cells.

  4. Add another Mix Onto element. This will add your cells + recovery media to the recovery media in the final Recovery Plate. Select Mix in Order as the Mix Mode and define the Transfer Volume

  5. Connect the Liquids from the Aliquot Recovery Media to the Liquids in Place.

  6. Connect Liquids from Add Recovery Media to Cells to Liquids to Add.

  7. Connect the Liquid Count from Add Recovery Media to Cells to Aliquot to Recovery Plate.

  8. Incubate the cultures for 1 hour at 37 °C.

    Your workflow should now look like this:

Mixing liquids based on sample metadata

We have now built your workflow to normalize your input liquids by concentration and execute a complex array of liquid mixing to perform a heat shock transformation protocol successfully.

In this section, we will:

  • Define antibiotic liquids to select the recovered transformations

  • Describe metadata values in the Define Plasmid DNA Table Editor

  • Add the required antibiotic to cultures as specified by the metadata

  1. Define two liquids you wish to add to your existing plate, based on metadata; for this example, we will define stock solutions of the commonly used antibiotics Kanamycin and Ampicillin.

  2. Modify the Define Plasmid DNA definition in the table editor. Add a metadata value called Antibiotic, using Add Subcomponent or Tag, then select Tag. Enter the values as required. Here we have defined A1-H1 as Kan and A2-H2 as Amp.

  3. Use Select to select Liquids from Incubate Cultures where the Antibiotic = Kan. Configure the selection criteria as shown below.

  4. Use Mix All to All mixture mode in Mix Onto to add 5ul of Kanamycin to all of the Selected Liquids.

  5. Repeat this process for the Ampicillin addition. For the second Selection connect the Unselected Liquids from the Kanamycin Select element into the Liquids input.

  6. Append the Mixed Liquids from the Kanamycin and Ampicillin addition together

  7. Append the Unselected Liquids from Ampicillin Selection.

  8. Incubate as desired.

Simulate the workflow

  1. Check that the device that you selected can follow the instructions that you prepared. To learn how, click here.

Preview the execution

  1. After you simulate the workflow, click View Simulation to open the simulation details.

  2. Open the Preview tab, then click through the steps to check that the instructions that Synthace has generated are correct.

    This complex workflow consists of the following steps:

    1. Normalization and aliquoting of the plasmid DNA.

    2. Aliquoting of the chassis cells for transformation.

    3. Mixing of DNA onto the cells.

    4. Heat-Shock prompts are generated for the user to perform the required incubations.

    5. Addition of recovery media to recover the heat-shocked cells.

    6. Incubation prompts for recovery.

    7. Antibiotic addition according to the Antibiotic metadata.

    8. Incubation prompt for selection of transformed cells.

Check your work:

To see what your finished workflow to this tutorial should look like simply navigate to the Tutorials and search for Tutorial 12: Build flexible, scalable workflows with the core elements.

Building flexible and scalable workflows

In order to make this workflow more flexible, we can add additional Select criteria for antibiotic addition. Say we want to run a new experiment and our input liquids changed to contain one plasmid which requires tetracycline. With our current workflow cultures with this plasmid would not be selected for Ampicillin or Kanamycin addition. However, we do not have elements to perform tetracycline addition. These samples would simply be missed from the selection step.

  1. Add Tetracycline to the Define Antibiotics element.

  2. Add and configure an additional Select and Mix Onto element for tetracycline addition in the same way we did for Kanamycin and Ampicillin. Set the Select element to match on Tet.

  3. Append the Mixed Liquids to the others.

  4. See below for the connection configuration.

If you simulate this workflow now you will see that the liquid handling has not changed, but if there were any Tet values in the Antibiotic metadata field the cultures transformed with this plasmid DNA would have Tetracycline added. You can see that although a Tetracycline Selection has been added to the workflow no tetracycline is being added and no tetracycline is present in the Reagents list on the Simulation Overview.

Ωx

You can test this by adding Tet to some of the plasmid DNA definitions. By building workflows in this way we can ensure they are flexible to any metadata requirements that your users might have. See the simulation in the workflow below. Compare this to the workflow above to see how Select is able to adapt the liquid handling behaviour to the change in metadata values.

​

Challenges

In this workflow some of the liquid transfers occur slightly out of order, for example the recovery media is aliquoted near the beginning of the workflow. Try to Synchronize the liquid handling steps to modify the execution order of this step.

Currently our Select elements require the metadata values (Kan or Amp) to match exactly. Can you modify the selection criteria to Select for common alternative values that might be defined by a user such as kan, kanamycin, kanamycin sulphate for example.

Hint: The Select element can be used to select Any matches specified in the metadata matching.

Related Articles
Mix all onto all
Normalize samples by concentration to a new plate
Cherry-pick samples based on concentration for normalization
Select liquids based on concentration and normalize to a new plate
Use of metadata for dilutions and mixing
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