Designing protocols - 9.5 - Designing a complete protocol

To design a protocol from scratch, you will need to:

• Define the plates and well arrays to be used
• Define the assets needed for the protocol
• Define the sequence of protocol steps
• Set asset or step properties where necessary
• Position the assets on the deck

The important points to remember are:

• firefly can only dispense into one plate or carry out one pipetting task at a time, so you cannot design a protocol with concurrent pipetting and dispensing steps.
• firefly and firefly+ grippers can continue to move items during a shake, heat/cool or incubate step, if you have selected 'Start and continue' behavior. The grippers cannot operate during dispensing, pipetting, loading or unloading steps as the heads will require access to the decks.
• firefly protocols must be linear, the protocol design doesn’t allow for conditional actions. Once a protocol is started, firefly will run it to the end unless the user intervenes and cancels it. If you want to automate a process which has different steps depending on an assay result, you will have to stop at the decision point.
• There are limitations on where labware can be placed to allow the dispensing and pipetting heads to access plates. Using the Deck wizard will ensure that you place your labware correctly.

You may find it helpful to design the main workflow of your protocol in a charting tool or sketch it by hand, before starting to build it up in firefly software.

firefly software is very versatile and allows you to use any design approach you wish when you start to build your protocol on the Design screen. You can start by defining the protocol steps, then add the required assets or start with the assets and then the steps to process them. There are no constraints on the order in which you build up a protocol.

Use Notes to organize long protocols into sections, you can also use Group to hide the details of completed process sequences and clarify the Steps view.

You can choose to view Plates, Steps or both when designing. Click the icon for the option which you find most useful.

You can also resize all the Design areas to show whatever is most convenient for your task by dragging the frame edges e.g., to maximise the Steps area as shown below. Blue lines mark the draggable frame edges.

The Editing a protocol section of this manual describes how to add and use plates, assets, and protocol steps in detail. When building them into a new protocol, there are various standard sequences for using them.

9.5.1 Pipetting

You will need to include these elements to carry out any pipetting task.

1. Loading tip sets or tip boxes and plates, using ‘Setup & loading’
2. A ‘load tips’ step to load the pipette head with tips.
3. A pipetting task (copy, mix or aspirate then dispense).
4. A ‘load tips’ step to remove the used tips, using the ‘unload only’ option. You can use the same tips repeatedly, or change them at whatever points in the protocol are appropriate. At the end of the protocol you must remove the last set of tips before you run another protocol or shutdown firefly. If there is liquid remaining in the tips, the software will prompt you to purge the tips before unloading.
5. A User interaction for unloading the plates and the used tips, unless they are to be
   unloaded after the protocol completes.

The pipette head requires enough space to access well arrays e.g., it cannot dispense into a deck location which has a tip stand to the left of it. Use the Auto fill deck (see section 9.5.6) to ensure that you place your labware appropriately.

9.5.2 Dispensing

You will need to include these elements to carry out any dispensing task.

1. Loading reservoirs, syringes and a destination plate using ‘Setup & loading’.
2. Either a fill step or separate aspirate and dispense steps. If there is liquid remaining in the syringes, the software will prompt you to purge the syringes before unloading.

If you will only be dispensing one or two reagents, and have plenty of material, you could use more than one reservoir for each and link the dispenses, and firefly will then dispense from multiple syringes in parallel and complete the step faster. This could be useful for larger volume dispense steps. The downside would be a larger reagent dead volume is needed, but this is recoverable at the end of the protocol.

9.5.3 Heating or chilling

9.5.3.i Plates

You will usually need to include these elements to use the plate thermal module.

1. A ‘Start and wait’ or ‘Start and continue’ step to start the plate thermal module heating or chilling, and set the required temperature. Using ‘Start and continue’ enables this to happen while other protocol steps are progressing.
2. A ‘Place on’ step to put the thermal block on the plate thermal module, unless you had loaded it there at the start of the protocol.
3. A ‘Place on’ step to put the plate on the thermal block, unless you had loaded the plate in that position at the start of the protocol.
4. One or more steps which will be carried out while the plate is being heated or cooled. This could be a pipetting step (which can be carried out while one of the plates is on the plate thermal module) or any other action not involving the plate.
5. A 'Move' or 'Take off' step to remove the plate from the plate thermal module.
6. A 'Stop' step, to turn off heating or chilling, once it is not needed.

Only the plate on the plate thermal module has active temperature control. You could have multiple thermal blocks to keep multiple plates chilled or heated, moving them around together and taking turns returning them to the plate thermal module for further heating or cooling. The thermal block will slow down the rate of temperature change of the plate contents to approximately 1°C per minute, and so can keep the plate temperature below 10°C for 5 minutes after you have cooled it on the plate thermal module to 4°C.

9.5.3.ii Reservoirs

You will need these elements to heat or cool reagents in reservoirs.

A ‘Start and wait’ or ‘Start and continue’ step to start the reservoir thermal module heating or chilling. Make this one of the first steps of the protocol if you want to load reagents and maintain their correct temperature, , or set the reservoir thermal plate temperature using the Decks screen. Use a 'Stop' step to end heating or cooling if it is no longer needed.

9.5.4 Incubate, using the thermal cycler

You will need these elements to use the ODTC. Before you incorporate an 'Incubate' step in your firefly protocol, you must have created the pre method and main method XML files in the ScriptEditor 3 software.

You can use lidded or unlidded plates on the ODTC. You can run an incubation step with the ODTC lid open or closed.

 

1.  Use an 'Incubate' step with Open lid behavior. 
2. Use a 'Move' step to transfer the plate to the thermal cycler. 
3. Use an 'Incubate' step with Close lid behavior, optionally. 
4. Use an 'Incubate' step to run the pre- method, or, alternatively use the 'Run both' Method
   Type to run the whole protocol. The purpose of the pre- method is to heat or cool the ODTC
   to the required temperature, so you may wish to run this using 'execute and wait' behavior,
   before loading the plate.  
5.  Use a second 'Incubate' step to run the main method. You can run multiple main methods
    sequentially, but to do this you must set the Post Heating property to true for all but the last
    method. 
   
   

6.      Use an 'Incubate' step with Open lid behavior, if you had closed the lid previously.

7.      Use another 'Move' step to remove the plate

If you are going to remove a lid from a plate after PCR, you may want to transfer it to a shelf
protector, to keep the firefly+ shelves clean. Note, the shelf protector appears as 'plate 2', as it is
defined as a plate type

 

 

Using hold temperature

You can utilise the ODTC's indefinite temperature hold function within a firefly protocol to keep a plate at a fixed temperature. This may be useful if you want to incubate successive plates at the same temperature, as loading and unloading plates does not stop an indefinite hold.

However, if you use this, when the firefly protocol completes, the ODTC will continue to run and hold the temperature, even after the plate has been removed from the ODTC.

So that it is not forgotten about, design the firefly protocol so that:

• If you are using the temperature hold in the middle of the firefly protocol, include a subsequent Incubate step with Behaviour set to Stop. This will cancel the indefinite hold.
• If you are using an indefinite hold at the end of the firefly protocol so that you can leave firefly running and come back for the plate at a convenient time, design your protocol with a User Interaction step to retrieve the plate. After that, include another Incubate step with Behaviour set to Stop as the last step. This way it will be clear that firefly and the ODTC are still running, and that the plate has been kept at a controlled temperature but needs to be unloaded.

9.5.5 Designing User interactions

When you design a user interaction, you are giving the firefly user instructions which will be displayed as a message on the screen so they must be clear and in the correct order.

You may want to include user interactions for unloading reagents and consumables used in your protocol, or you may want to complete the protocol and then use your lab procedures for emptying and cleaning firefly

• Use the correct Rest state (see section 9.4.20) to enable the user to access specific areas safely.
• Use the unloading instructions (see section 6.6) as a prompt for your user interactions and SOPs.
• Write a series of short user interaction steps rather than one detailed complex instruction, if the user has multiple tasks to carry out. That way they can confirm when they have finished each one.
• If the user is going to need labware or reagents which are not loaded in firefly, create a User interaction early in the protocol to confirm that those items are available and ready for use. This is particularly important if there are time critical steps in the protocol which must not be delayed.
• Think ahead: if the plate will need to be transferred to another instrument, does it need time to set up or to reach a temperature setpoint? If so, you should have a User interaction to get that instrument ready at an earlier stage in the protocol, so the user is preparing it while firefly is performing other steps.
• You could give the user conditional instructions, e.g., the first user interaction is to perform an assay and the second is to use the results to determine how much diluent to add to a sample.
• If there is anything particularly complex, the User interaction could direct the user to another method document in the lab, to display charts, diagrams or more detailed instructions than can be shown on firefly’s tablet screen.

9.5.6 Stacking Assets

You use the pipetting head grippers to stack plates on top of other assets, using 'Move' or ‘Place on’ to create a stack, and 'Move' or ‘Take off’ to separate the items. Some items, like the plate thermal module, can only be base items.

To move stacked assets together, drag and drop the bottom item from Assets to Steps, when designing. The graphic will show that the two assets are moved together.

Using stacks, you can keep a plate on a magnetic block or thermal block while pipetting or dispensing.

You can only use combinations of lids and plates or plates and base items which have a stack
definition. Use Community to download stack definitions. If you cannot find what you need, 
contact reliance.

9.5.7 Setting up the Decks

Once you have finalized the labware your protocol will require, open the Deck screen to set up loading firefly.

You can set the initial temperature of the reservoir or plate thermal blocks.

Use the Auto Deck Fill (highlighted above) to automatically select the layout for your assets.

You can drag and drop your assets onto deck locations. If you do this, check the Errors panel on
the Steps view afterwards, in case you need to revise your layout. There is a Reset Decks function which empties the entire deck, to start again if you need to. To remove a single asset, right click on it and select 'Remove'. You can then drag it to a new location.

If you are not using Auto Deck fill, the allowed positions for labware loading are shown below.

EZL head

ATL head

9.5.8 Using firefly+

The Auto Deck Fill function will use the shelves in firefly+ to place suitable labware. There are
height restrictions (shown in mm below) for the labware which you can place on each shelf type.

The overall allowed storage positions are:
If the shelves are in use, the firefly+ grippers need a clear deck location to place labware, before it is used in a protocol. This can be U2, U3, L2 or L3. You may need to modify your deck layout manually to clear one of these deck locations if you see error messages when you attempt to run your protocol.

Ideally try and keep all 4 of these locations (or as many as are practical for your materials) empty, when setting up the decks for your protocol, as it will make protocol execution more efficient. 

9.5.9 Getting feedback from the Errors panel

Check the Errors panel as you are designing your protocol: it will notify you of volumetric inconsistencies or missing steps such as using tips without a prior Load Tips step. There are 3 levels of severity:

1. Errors are the most serious problems i.e., when you have not defined a step correctly. If there are Errors, your protocol cannot be executed.
2. Warnings will not prevent firefly executing your protocol, but they alert you to significant problems i.e., when cross contamination may occur.
3. Information messages alert you to minor issues or possible improvements.

Everywhere you see ‘{xxx}’ there will be the name of an asset, a plate, a well volume or other
specific item in the actual error messages.

  Clicking on the associated step number will highlight the step in the step area.

Resolve all errors to enable the protocol to run.

Errors

Well arrays

Tips & Pipetting

Syringes & dispensing

Pause

Thermal modules

Shaker

ODTC

Stacks

Collision Detection

Height Restrictions

firefly+ 

Error or warning depending on asset

Warnings

Information

9.5.10 Collision detection

As you design your protocol, firefly will warn you about collisions, where labware will obstruct the movement of the pipetting head e.g., if you are loading tips from a tip box when there is a plate on the thermal module.

The error message will suggest modifications to solve the collision issue, but you may not want to implement them. If you use Auto Fill Decks while you have an unsolved collision error, you will be warned that you need to place your labware manually.

As you cannot execute a protocol which has errors, you can try and solve the collision problem by:

• manually dragging and repositioning the labware which will collide, particularly if you have
  empty positions available on the decks
• revise the order of protocol steps other than the suggested modification if you have scope to
  do this
• use the deck appropriate riser to lift the lowest plate and so raise the working height of the head, avoiding collisions

Keep checking the Errors panel as you make changes, to see whether you have resolved the error.

Next Article - Designing protocols - 9.6 - Testing your protocol in simulation