Designing protocols - 9.4 - Editing steps

When editing protocol steps, check the Errors panel on the right of the screen, in case your
changes cause problems elsewhere in the protocol which will need you to make changes to other
steps e.g. to load larger reagent volumes.

9.4.1 Adding steps

To add a step at the end of a protocol, click the Add step ‘+’ icon, use the Protocol menu item or click the ‘+’ icon below the Steps list on the right of the screen.

Select the action you want to add from the 'Add Step' menu or 'Add Step' pop-up form.

You can copy and paste one or more steps should you need them repeatedly in a protocol. 

9.4.2 Inserting steps

To insert a step part way through a protocol:

1. Select the step which will immediately follow the new addition.
2. Right click and select ‘Insert’ from the popup menu to bring up the protocol steps menu, or select the main Protocol menu in the toolbar, then select ‘Insert Step’ from the dropdown menu.
3. Select the specific function you wish to add from the insert menu.

Alternatively, you can use the insert function on the Steps panel. Again, select the step which will
immediately follow the new addition, then use the Insert function.

9.4.3 Grouping steps

If you have a long protocol, or one where the same steps and methods are used repeatedly, you can group them into a single Group. Select the steps, then click Group, on the left.

You will then see a single icon for all the grouped steps, which you can move, cut, copy and paste to use it repeatedly in your protocol.

Give your group a descriptive name as you will not immediately see the steps contained in the group in the Steps view in Execute.

You will see the individual steps when you execute (see section 6.5) the protocol.

You can ungroup them again by clicking the ungroup icon on the right. You can do this to see the steps contained in the group.

You do not need to use Ungroup to edit the steps. Double-click the group icon.

This opens the steps within the group.

If you have nested groups, you could then open the nested group in the same way (you can only have one group level at a time open).

The breadcrumbs bar will always show you which group you have open and where it sits within the protocol. 

To exit a group and return to an 'outer' level, click the level you want to move to in the breadcrumbs bar (highlighted in red - this will exit to the main protocol level).

9.4.4 Repeating steps

To repeat a step exactly, right click on it and select Copy from the right click menu, or use the Ctrl-C shortcut. Right click again and select Paste (or use the Ctrl-V shortcut) and the step will be added to the end of the protocol, with all Properties unchanged. 

To repeat a sequence of steps, make them into a group (see section 9.4.3) and set the group’s Cycles property to the number of repetitions you require.

To repeat a sequence at intervals throughout you protocol, create a Group for those steps and use Copy and Paste to use them repeatedly.

9.4.5 Re-ordering steps

To change the order in which steps execute, select the step you want to move, and drag it to its correct position. You may find it useful to adjust the Steps zoom level first, to get the view you need.

You may find it easiest to use Group before moving multiple steps, and then ungroup them after using drag and drop.

9.4.6 Deleting steps

To delete a step, either select it in the Steps or the Steps list at the right of the screen, then click on the bin below the Steps list, use the keyboard Delete key or Delete in the right click menu.

IMPORTANT: There is no undo function to reverse a deletion.

9.4.7 Pipetting Head: Load / Change tips

Load / Change tips enables you to switch to clean pipetting tips. You will need to include this function twice in any protocol which will carry out pipetting operations, to load tips at the start and to unload them at the end of the protocol. Add a Tip Set as an asset, and then drag it to the Load / Change tips step.

You can use multiple tip sets in a protocol, to keep using new tips, but you will need to load sufficient tip sets for each change of tips.

If you need more tip sets than you have space to load at the start of the protocol, use User Interactions to remove used tip sets and replenish new ones. A maximum of 8 tip sets can be defined and used in a protocol. For this reason ensure that the fresh tips which are loaded when the tips are replenished, are of the same type as the tips that are removed from the deck.

At the end of a protocol, you must unload the final set of tips. The protocol will not execute if tips remain on the pipetting head at the end of the protocol. 

Use the ‘Load only’ behavior when there are no tips on the head i.e. the first tip loading operation, and ‘Unload only’ at the end of the protocol. The Load/Change tips icon is modified, depending on the behavior selected; this makes it easier to keep track of what tips are available.

9.4.8 Pipetting Head: Copy

Copy combines aspirate and dispense pipetting functions to copy a well array to a second well array, which can be in the same plate or in a second plate. The well arrays must be the same size and shape as shown below.

You can copy between 96 and 384 well plates, if you use an appropriate tiling pattern in the 384 well plate.

If you select a liquid class, other properties will automatically be set to suitable values, though you can edit these if you wish.

IMPORTANT: firefly software does not validate Tip Offset limits. Use these with caution. Incorrect values could lead to crashes or out of range execution errors. The range is mostly determined by the plate definition but there are absolute limits. For a 96-well plate +/-4.5 mm (half the well pitch) from the well center would be the absolute limit and +/-2.25 mm for a 384 well plate. 

Aspirate

Tip Tracking

Tip Touch

Dispense

Tip Tracking

Tip Touch

7.4.9 Pipetting Head: Aspirate

Aspirate is a simple function to aspirate reagent from a plate well into a pipette tip.

You can aspirate from a single plate or perform multiple aspirate steps into the same tips from different wells, so long as there is sufficient volume. You can use the aspirate and dispense functions to pipette between well arrays which are not possible when using Copy.

firefly software will warn you in the Errors panel if you have selected volumes which are
incompatible with the tips or wells used.

You can adjust multiple parameters for this step, but the pre-set default values should work for
most reagents. If you select a liquid class, other properties will automatically be set to suitable
values, though you can edit these if you wish. Use the Advanced properties if you have reagents
which are particularly demanding, or for which you already know the optimal pipetting
parameters in detail.

IMPORTANT: firefly software does not validate Tip Offset limits. Use these with caution. Incorrect values could lead to crashes or out of range execution errors. The range is mostly determined by the plate definition but there are absolute limits. For a 96-well plate +/-4.5 mm (half the well pitch) from the well center would be the absolute limit and +/-2.25 mm for a 384 well plate. 

Aspirate

Tip Tracking

Tip touch

Tip tracking is a facility to move the tip lower into the well as more liquid is aspirated. By keeping the tip a set distance below the surface of the liquid, it is possible to ensure that the sample is aspirated from e.g. a particular layer, if the well contents have been separated.

9.4.10 Pipetting Head: Dispense

Dispense is a simple function to pipette from a tip into a well array.

You do not need to select a source; firefly will use the reagent which has already been aspirated into the pipette head.

Selecting a dispense step without a previous aspirate step will cause an error in the protocol.You can adjust multiple parameters for this step, but the pre-set default values should work for most reagents.

Use the Advanced properties if you have reagents which are particularly demanding, or for which you already know the optimal dispense parameters in detail.

IMPORTANT: firefly software does not validate Tip Offset limits. Use these with caution. Incorrect values could lead to crashes or out of range execution errors. The range is mostly determined by the plate definition but there are absolute limits. For a 96-well plate +/-4.5 mm (half the well pitch) from the well center would be the absolute limit and +/-2.25 mm for a 384 well plate. 

Dispense

Tip Touch

Tip Tracking

9.4.11 Pipetting Head: Mix

Mix aspirates then dispenses repeatedly, to mix the contents of a well. You can set the aspirate and dispense heights within the well to enhance the mixing of reagents in layers.

This step includes an additional aspirate and dispense at the end of the mix, this can be performed at a different speed or height in order to ensure clean tips and prevent tips dripping or being dirty when leaving the plate.

You can adjust multiple parameters for this step, but the pre-set default values should work for most reagents.

IMPORTANT: firefly software does not validate Tip Offset limits. Use these with caution. Incorrect values could lead to crashes or out of range execution errors. The range is mostly determined by the plate definition but there are absolute limits. For a 96-well plate +/-4.5 mm (half the well pitch) from the well center would be the absolute limit and +/-2.25 mm for a 384 well plate. 

Pre-mix

Mix

Aspirate

Dispense

If you have the plate thermal module behavior as ‘Start and continue’, your plate will be kept at the set temperature while you carry out subsequent steps. You can use a Pause step if you want to continue heating or cooling for a defined period, or if you don’t need any concurrent actions.

Use another Heat / Cool step if you want to modify the temperature while the plate is on the plate thermal module.

Use Move or Take Off to remove the plate, or select the thermal block to remove and the plate together as a stacked object.

Use a Heat / Cool step with ‘Stop’ behaviour to stop the plate thermal module from heating or cooling once you finish using it. This will minimise the heat emissions within firefly and help to keep the internal temperature stable. If you have heated the plate thermal module, this will cool it as soon as possible, making user access to the basement safer.

9.4.23 Labware & Process Modules: Shake

To use the Shaker Module, you must first add it as an Asset, then use Move or Place On to move the plate to it.

Use Shake to control the plate shaking process by setting the behavior properties: 

• Choose ‘Start and continue’ to have the protocol continue with the next step while the Shaker is shaking.
• If you want shaking to continue for the duration of the subsequent step or steps and only stop when that is complete, set the Duration to a much longer period than those steps will require, then include a Shake step with Stop as the behavior after the last of those steps.
• Choose ‘Start and wait’, to have firefly do nothing until the shake duration is completed.

Then use Move or Take Off to remove the plate from the shaking module.

9.4.24 User interaction

Create a User interaction step whenever you need the user to add or remove materials from firefly, to remove the plate for processing on a different instrument e.g. a thermocycler or any other manual task.

When the protocol runs, the Description will be shown to the user as instructions. When they click OK, they will be confirming that they have completed all the manual tasks and that firefly should continue running the protocol. You might want to include that in your instructions.

Use the rest state property to enable safe access to firefly. As it is not possible to easily access all areas of the instrument at the same time, setting this property will ensure decks and heads are moved clear of the labware requiring user interaction.

• If you need to access both the reservoirs and the upper deck, create two consecutive user interaction steps, one for each area, setting the rest state for each area in turn. 
• If you do not want the heads moved, or any disruption to firefly hardware during the user interaction, select ‘Do nothing’. This may make access more awkward and you might want to warn the user about that in the User Interaction instructions.

9.4.25 Notes

Notes appear in protocol design views as Post-It notes. Use them to give information to the user that does not interrupt the firefly workflow.

When executing a protocol, notes are shown with a yellow background in the Steps.

9.4.26 Pausing

Pause will put firefly into an inactive state e.g. while a plate is cooling on the plate thermal module.

You can use the rest state to move heads away from plates e.g. by homing them, if you are concerned that reagents might drip into the plates during the pause.

You do not need to use Pause for a user interaction i.e. to remove the plate from firefly. The instrument will pause until the user clicks ‘OK’ on the user interaction instruction to confirm that they have completed it.

Next Article - Designing protocols - 9.5 - Designing a complete protocol