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.

Add step

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.

add insert step

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.

Group & ungroup

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.

Group stepGive 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.

Property Form Notes
Description Free text Use this to give your group a meaningful name
Color Select from pop-up form Use to identify and differentiate groups
Cycles Numeric 1 or more iterations of the grouped steps

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.

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This opens the steps within the group.

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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).

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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).

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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.

delete step

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. 

Property Form Notes
Description Free text  
Source [auto filled from the drag and drop operation, not editable]  
Behaviour Select from dropdown list Options are: Unload then load, Load only, Unload only

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.

Tip load icon

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.

Copy pipettingYou 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. 

Property Form Notes
Description Free text  
Source Auto filled from the drag and drop operation, but can be edited from dropdown list The location from which liquid is aspirated.
Destination Auto filled from the drag and drop operation, but can be edited from dropdown list The location to which liquid is dispensed.
Volume (μL) Numeric The volume of liquid transferred per well, from the source wells to the destination wells.
Liquid Class Select from dropdown list Select from all installed liquid classes

Aspirate

Speed (μL/s) Numeric The speed at which liquid is aspirated into the tip.
Blowout volume (μL) Numeric A volume of air that is aspirated before the liquid.
Air transport volume (μL) Numeric A volume of air that is aspirated after the liquid.
Pause (s) Numeric Delay immediately after liquid is aspirated into the tips.
Extraction speed (mm/s) Numeric Speed at which the tips move upwards after liquid has been aspirated into the tips.
Tip offset (mm) Numeric The distance the tip is offset from the centre of the well for the aspiration.
Tip move speed (mm/s) Numeric Speed at which the tips move to the start height for the aspiration.
Keep tips in wells Tick box Keeps the tips at the aspirate end height after liquid has been aspirated into the tips. No tip touch will be performed.

Tip Tracking

Auto tracking Tick box Automated z-movement to maintain tips beneath the liquid for the aspiration.
Tip Liquid Offset (mm) Numeric Distance of the tips below the liquid surface when auto tracking is used. Set to zero if auto tracking is not used.
Start height (%) Numeric Height in the well at which the aspirate will start when auto tracking is not used. The well graphic updates with your changed value.
End height (%) Numeric Height in the well at which the aspirate will finish when auto tracking is not used. The well graphic updates with your changed value.

Tip Touch

Method Select from dropdown list Position the tips will touch in the well after the aspiration. Specify the side of the well or surface of the liquid.
Height (%) Numeric Height from the top of the well at which the tip touch is performed. The well graphic updates with your changed value.
Dwell period (ms) Numeric Duration for which the tips will remain at the tip touch position
Force Select from dropdown list The speed at which the tip touch is performed.

Dispense

Speed (μL/s) Numeric The speed at which liquid is dispensed from the tips.
Blowout volume (μL) Numeric Volume of air to dispense from the tips after the liquid.
Air transport volume (μL) Numeric Volume of air to dispense from the tips before the liquid.
Pause (s) Numeric Delay immediately after liquid is dispensed from the tips.
Extraction speed Numeric Speed at which the tips move upwards after liquid has been dispensed from the tips.
Tip offset (mm) Numeric The distance the tip is offset from the centre of the well for the dispense.
Tip move speed (mm/s) Numeric Speed at which the tips move to the dispense start height.
Keep tips in wells Tick box Keeps the tips at the dispense end height after liquid has been dispensed from the tips. No tip touch will be performed.

Tip Tracking

Auto tracking Tick box Automated z-movement to maintain tips beneath the liquid for the dispense
Tip tracking offset (mm) Numeric Distance of the tips below the liquid surface when auto tracking is used. Set to zero if auto tracking is not used.
Start height (%) Numeric Height in the well at which the dispense will start when auto tracking is not used. The well graphic updates with your changed value.
End height (%) Numeric Height in the well at which the dispense will finish when auto tracking is not used. The well graphic updates with your changed value.

Tip Touch

Method Select from dropdown list Position the tips will touch in the well after the dispense. Specify the side of the well or surface of the liquid.
Height (%) Numeric Height from the top of the well at which the tip touch is performed. The well graphic updates with your changed value.
Dwell period (ms) Numeric Duration for which the tips will remain at the tip touch position.
Force Select from dropdown list
The speed at which the tip touch is performed.

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.

Aspirate & dispense pipettingfirefly 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. 

Property Form Notes
Description  Free text  
Source Auto filled from the drag
and drop operation, but
can be edited from
dropdown list
The location from which liquid is aspirated.
Volume (µL/tip) Numeric / Variable The volume of liquid transferred per well,
from the source wells to the destination wells.
Liquid class Select from dropdown list Select from all installed liquid classes

Aspirate

Speed (µL/s) Numeric The speed at which liquid is aspirated into the tip. 
Blowout volume (µL) Numeric A volume of air that is aspirated before the liquid. 
Air transport volume (µL) Numeric A volume of air that is aspirated after the liquid.
Pause (s) Numeric Delay immediately after liquid is aspirated into the tips.
Extraction speed (mm/s) Numeric Speed at which the tips move upwards after liquid has been aspirated into the tips.
Tip offset (mm) Numeric The distance the tip is offset from the centre of the well for the aspiration. 
Tip move speed (mm/s) Numeric Speed at which the tips move to the start height for the aspiration.
Keep tips in wells Tick Box Keeps the tips at the aspirate end height after liquid has been aspirated into the tips.
No tip touch will be performed. 

Tip Tracking

Auto tracking Tick Box Automated z-movement to maintain tips beneath the liquid for the aspiration. 
Tip liquid offset (mm) Numeric Distance of the tips below the liquid surface when auto tracking is used. Set to zero if auto tracking is not used. 
Start height (%)  Numeric Height in the well at which the aspirate will start when auto tracking is not used. The well graphic updates with your changed value. 
End height (%) Numeric Height in the well at which the aspirate will finish when auto tracking is not used. The well graphic updates with your changed value. 

Tip touch

Method Select from dropdown list Position the tips will touch in the well after the aspiration. Specify the side of the well or surface of the liquid.
Height (%) Numeric Height from the top of the well at which the tip touch is performed. The well graphic updates with your changed value.
Dwell period (ms) Numeric Duration for which the tips will remain at the tip touch position.
Force Select from dropdown list The speed at which the tip touch is performed. 

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. 

Property Form Notes
Description Free text  
Destination Auto filled from the drag and drop operation, but can be edited from dropdown list  
Volume (μL/tip) Numeric The volume of liquid transferred per well, from the source wells to the destination wells.
Liquid class Select from dropdown list  Select from all installed liquid classes

Dispense

Speed (μL/s) Numeric The speed at which liquid is aspirated into the tip.
Blowout volume (μL) Numeric A volume of air that is aspirated before the liquid.
Air transport volume (μL) Numeric Volume of air to dispense from the tips before the liquid.
Pause (s) Numeric Delay immediately after liquid is aspirated into the tips.
Extraction speed Numeric Speed at which the tips move upwards after liquid has been dispensed from the tips.
Tip offset (mm) Numeric The distance the tip is offset from the centre of the well for the dispense.
Tip move speed (mm/s) Numeric Speed at which the tips move to the dispense start height.
Keep tips in wells Tick box Keeps the tips at the aspirate end height after liquid has been aspirated into the tips. No tip touch will be performed.

Tip Touch

Auto tracking Select from dropdown list Position the tips will touch in the well after the dispense. Specify the side of the well or surface of the liquid.
Tip Liquid Offset (mm) Numeric Height from the top of the well at which the tip touch is performed. The well graphic updates with your changed value.
Start height (%) Numeric Duration for which the tips will remain at the tip touch position.
End height (%) Select from dropdown list The speed at which the tip touch is performed.

Tip Tracking

Method Select from dropdown list Position the tips will touch in the well after the dispense. Specify the side of the well or surface of the liquid.
Height (%) Numeric Height from the top of the well at which the tip touch is performed. The well graphic updates with your changed value.
Dwell period (ms) Numeric Duration for which the tips will remain at the tip touch position.
Force Select from dropdown list The speed at which the tip touch is performed.

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. 

Property Form Notes
Description Free text  
Target Auto filled from the drag and drop operation, but can be edited from dropdown list  
Keep tips in wells Tick box Keeps the tips at the dispense end height after liquid has been dispensed from the tips. No tip touch will be performed.
Extraction speed (mm/s) Numeric Speed at which the tips move downwards to the mix start height and upwards on completion of the mix.

Pre-mix

Blowout volume (μL) Numeric Volume of air aspirated into the tip before the mix.

Mix

Mix volume (μL) Numeric Volume to aspirate and dispense for the mix.
Mix cycles Numeric Number of times the mix volume is aspirated then dispensed.
Aspirate height (%) Numeric Height from the top of the well that the mix aspiration will start. The well graphic updates with your changed value.
Dispense height (%) Numeric Height from the top of the well that the mix dispense will start. The well graphic updates with your changed value.
Tracking Tick box If not selected, the mix aspirate is performed at the aspirate height and the mix dispense is performed at the dispense height. If selected, the mix aspirate starts at aspirate height and ends at the dispense height and the mix dispense starts at the dispense height and ends at the aspirate height.
Tip tracking offset (mm) Numeric This is a legacy parameter and should not be used.

Aspirate

Speed (μL/s) Numeric Speed at which liquid is aspirated into the tips.
Pause (s) Numeric Delay immediately after liquid is aspirated into the tips.
Tip offset (mm) Numeric The distance the tip is offset from the centre of the well for the aspiration.

Dispense

Speed (μL/s) Numeric Speed at which liquid is dispensed from the tips.
Pause (s) Numeric Delay immediately after liquid has been dispensed from the tips.
Tip offset (mm) Numeric The distance the tip is offset from the centre of the well for the dispense.

Post mix

Auto tracking Tick box Automated z-movement to maintain tips beneath the liquid for the final aspiration and/or dispense after the mix.
Tip Liquid Offset (mm Numeric Distance of the tips below the liquid surface when auto tracking is used. Set to zero if auto tracking is not used.

Aspirate

Perform aspirate Tick box The post mix aspirate is optional.
Volume (μL) Numeric Volume of liquid transferred per well.
Speed (μL/s) Numeric The speed at which liquid is aspirated into the tip.
Air transport volume (μL) Numeric A volume of air that is aspirated after the liquid.
Pause (s) Numeric Delay immediately after liquid is aspirated into the tips.
Start height (%) Numeric Height in the well at which the aspirate will start when auto tracking is not used. The well graphic updates with your changed value.
End height (%) Numeric Height in the well at which the aspirate will finish when auto tracking is not used. The well graphic updates with your changed value.
Tip offset (mm) Numeric The distance the tip is offset from the centre of the well for the aspiration.

Dispense

Perform dispense Tick box The post mix dispense is optional
Volume (μL) Numeric Volume of liquid to dispense
Speed (μL/s) Numeric Speed at which liquid is dispensed from the tips
Air transport volume (μL) Numeric Volume of air to dispense from the tips before the liquid
Blowout volume (μL) Numeric Volume of air to dispense from the tips after the liquid
Pause (s) Numeric Delay immediately after liquid is dispensed from the tips.
Start height (%) Numeric Height in the well at which the dispense will start when auto tracking is not used. The well graphic updates with your changed value.
End height (%) Numeric Height in the well at which the dispense will finish when auto tracking is not use. The well graphic updates with your changed value.
Tip offset (mm) Numeric Distance of the tips below the liquid surface when auto tracking is used. Set to zero if auto tracking is not used.

Tip touch

Method Select from dropdown list Position the tips will touch in the well after the dispense. Specify the side of the well or surface of the liquid.
Height (%) Numeric Height from the top of the well at which the tip touch is performed. The well graphic updates with your changed value.
Dwell period (ms) Numeric Duration for which the tips will remain at the tip touch position.
Force Select from dropdown list The speed at which the tip touch is performed.

9.4.12 Pipetting Head: Purge

Purge is used to empty remaining reagent from tips e.g. before they are unloaded.  

Use the Advanced Tip Tracking and Tip Touch functions if you want to be sure the tips are clean after purging, with no droplets on the tips. 

Property Form Notes
Description Free text  
Destination Auto filled from the drag and drop operation but can be edited from dropdown list  
Purge    
Purge speed (µL/s) Numeric Speed at which liquid is purged from the tips
Purge pause (s) Numeric  
Tip tracking    
Auto tracking Tick box Automated z-movement to maintain tips beneath the liquid
Tip liquid offset (mm) Numeric Distance of the tips below the liquid surface when auto tracking is used. Set to zero if auto tracking is not used.
Start height (%) Numeric Height in the well at which the purge will start when auto tracking is not used. The well graphic updates with your changed value.
End height (%) Numeric Height in the well at which the purge will finish when auto tracking is not used. The well graphic updates with your changed value.
Tip touch    
Method Select from dropdown list Position the tips will touch in the well after the purge. Specify the side of the well or surface of the liquid. 
Height (%) Numeric Height from the top of the well at which the tip touch is performed. The well graphic updates with your changed value.
Dwell period (ms) Numeric Duration for which the tips will remain at the tip touch position.
Force Select from dropdown list The speed at which the tip touch is performed.

 

9.4.12 Syringe Head: Aspirate

Aspirate is a simple function to aspirate reagent from a reservoir into a syringe.

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.

Property Form Notes
Description Free text  
Source [auto filled from the drag and drop operation, not editable]   
Volume (μL) Numeric Volume of liquid aspirated.
Preset Select from dropdown list Options for specific aspirate tasks

Aspirate

Speed (mm/s) Numeric Aspirate speed of the liquid
Safeguard volume (μL) Numeric Additional volume aspirated into the syringe which remains in the syringe after the dispense. When using separate Dispense Head Aspirate and Dispense steps do not alter the default 40µL safeguard volume for the Aspirate to avoid a volume mismatch software error with the Dispense step.

Pre wet

Use Pre Wet Tick box Aspirate then dispense liquid into the syringe to wet the syringe body and plunger. It is required before the initial aspiration of liquid into the syringe.
Repetitions Numeric Number of aspirate/dispense cycles to perform for the pre-wet.
Speed (mm/s) Numeric The speed the piston rod moves the plunger up during the pre-wet. This will change the speed the liquid is aspirated into the syringe.
Pause (ms) Numeric Time delay between each aspirate-dispense cycle of the pre-wet. It is not applied after the final repetition of the pre-wet.
Shot distance (mm) Numeric The distance the piston rod is forced down by the solenoid for the shot of the pre-wet. Must be less than the up-down distance.
Shot duration (ms) Numeric Duration the solenoid is energised to execute the shot of the pre-wet.
Updown distance (mm) Numeric Distance the plunger moves up/down for the pre-wet.

Pre dispense 1

Use Pre Dispense 1 Tick box Priming dispense(s) performed prior to dispensing to the destination plate.
Volume (μL) Numeric Volume to dispense per repetition of the pre-dispense.
Repetitions Numeric Number of times to dispense the pre-dispense volume.
Suckback threshold (μL) Numeric The maximum dispense volume to which suckback is applied. For solenoid dispenses only.
Pre Dispense pause (ms) Numeric The pause before each pre-dispense repetition. For solenoid dispenses only.
Post Dispense pause (ms) Numeric The pause after each pre-dispense repetition. For solenoid dispenses only.

Pre dispense 2

Use Pre Dispense 2 Tick box Priming dispense(s) performed prior to dispensing to the destination plate.
Volume (μL) Numeric Volume to dispense per repetition of the pre-dispense.
Repetitions Numeric Number of times to dispense the pre-dispense volume.
Suckback threshold (μL) Numeric The maximum dispense volume to which suckback is applied. For solenoid dispenses only.
Pre Dispense pause (ms) Numeric The pause before each pre-dispense repetition. For solenoid dispenses only.
Post Dispense pause (ms) Numeric The pause after each pre-dispense repetition. For solenoid dispenses only.

9.4.13 Syringe Head: Dispense

Dispense is a simple function to dispense from one or more syringes into a well array. Select a reservoir for the source; firefly understands to use the syringe filled from that reservoir, and if there are multiple reservoirs filled with the same reagent, to use all the syringes for faster parallel dispensing.

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.

Property Form Notes
Description Free text  
Source Auto filled from the drag and drop operation, but can be edited from dropdown list  
Destination Auto filled from the drag and drop operation, but can be edited from dropdown list  
Volume (μL) Numeric  
Preset Select from dropdown list Options for specific tasks

Dispense

Syringe speed (mm/s) Numeric The speed at which the piston rod moves downwards for solenoid dispenses.
Syringe ramp Select from dropdown list Acceleration profile of the piston rod for a solenoid dispense.
Max Shot Volume (μL) Numeric Maximum volume of a droplet used during a solenoid dispense.
Single Shot Min Volume (μL) Numeric Minimum volume of a droplet used during a solenoid dispense.
Multi Shot Min Volume (μL) Numeric Minimum volume of a droplet used during a multi shot solenoid dispense
Pre Dispense Pause (ms) Numeric The pause before each dispense. For solenoid dispenses only.
Post Dispense Pause (ms) Numeric The pause after each dispense. For solenoid dispenses only.
Syringe Z Offset (mm)  Numeric Z offset when putting syringe into well automatically does an up-down move in between dispenses.
Syringe Offset (mm)  Numeric The distance the syringe is offset from the centre of the well for the dispense.

Direct drive dispense

Move Speed (mm/s) Numeric The speed at which the piston rod moves downwards for direct-drive dispenses
Inter Dispense pause (ms) Numeric  
Threshold (μL) Numeric The volume above which direct-drive dispensing is enabled
Auto Finishing Shot Tick box This step is optional, though included by default for direct drive dispensing.
Finishing Shot Volume (µL) Numeric  

9.4.14 Syringe Head: Fill

Fill is a single step function to aspirate from a reservoir and dispense into a well array. Drag and drop the required assets onto the step and check there are no volumetric errors.

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

Property Form Notes
Description Free text  
Source Auto filled from the drag and drop operation, but can be edited from dropdown list  
Destination Auto filled from the drag and drop operation, but can be edited from dropdown list  
Volume (μL) Numeric Volume of liquid aspirated.
Preset Select from dropdown list Options for specific tasks

Aspirate

Speed (mm/s) Numeric Aspirate speed of the liquid
Safeguard volume (μL) Numeric Additional volume aspirated into the syringe which remains in the syringe after the dispense.

Pre wet

Use Pre Wet Tick box Aspirate then dispense liquid into the syringe to wet the syringe body and plunger. It is required before the initial aspiration of liquid into the syringe.
Repetitions Numeric Number of aspirate/dispense cycles to perform for the pre-wet.
Speed (mm/s) Numeric The speed the piston rod moves the plunger up during the pre-wet. This will change the speed the liquid is aspirated into the syringe.
Pause (ms) Numeric Time delay between each aspirate-dispense cycle of the pre-wet. It is not applied after the final repetition of the pre-wet.
Shot distance (mm) Numeric The distance the piston rod is forced down by the solenoid for the shot of the pre-wet. Must be less than the up-down distance.
Shot duration (ms) Numeric Duration the solenoid is energised to execute the shot of the pre-wet.
Updown distance (mm) Numeric Distance the plunger moves up/down for the pre-wet.

Pre dispense 1

Use Pre Dispense 1 Tick box Priming dispense(s) performed prior to dispensing to the destination plate.
Volume (μL) Numeric Volume to dispense per repetition of the pre-dispense.
Repetitions Numeric Number of times to dispense the pre-dispense volume.
Suckback threshold (μL) Numeric The maximum dispense volume to which suckback is applied. For solenoid dispenses only.
Pre Dispense pause (ms) Numeric The pause before each pre-dispense repetition. For solenoid dispenses only.
Post Dispense pause (ms) Numeric The pause after each pre-dispense repetition. For solenoid dispenses only.

Pre dispense 2

Use Pre Dispense 1 Tick box Priming dispense(s) performed prior to dispensing to the destination plate.
Volume (μL) Numeric Volume to dispense per repetition of the pre-dispense.
Repetitions Numeric Number of times to dispense the pre-dispense volume.
Suckback threshold (μL) Numeric The maximum dispense volume to which suckback is applied. For solenoid dispenses only.
Pre Dispense pause (ms) Numeric The pause before each pre-dispense repetition. For solenoid dispenses only.
Post Dispense pause (ms) Numeric The pause after each pre-dispense repetition. For solenoid dispenses only.

Dispense

Syringe speed (mm/s) Numeric The speed at which the piston rod moves downwards for solenoid dispenses.
Syringe ramp Select from dropdown list Acceleration profile of the piston rod for a solenoid dispense.
Max Shot Volume (μL) Numeric Maximum volume of a droplet used during a solenoid dispense.
Single Shot Min Volume (μL) Numeric Minimum volume of a droplet used during a solenoid dispense.
Multi Shot Min Volume (μL) Numeric Minimum volume of a droplet used during a multi shot solenoid dispense.
Pre Dispense Pause (ms) Numeric The pause before each dispense.For solenoid dispenses only.
Post Dispense Pause (ms) Numeric The pause after each dispense.For solenoid dispenses only.
Syringe Z Offset (mm)  Numeric Z offset when putting syringe into well automatically does an up-down move in between dispenses.
Syringe Offset (mm)  Numeric The distance the syringe is offset from the centre of the well for the dispense.

Direct drive dispense

Move Speed (mm/s) Numeric The speed at which the piston rod moves downwards for direct-drive dispenses.
Inter Dispense pause (ms) Numeric  
Threshold (μL) Numeric The volume above which direct-drive dispensing is enabled.
Auto Finishing Shot  Tick box This step is optional, though included by default for direct drive dispensing.
Finishing Shot Volume (µL) Numeric  

9.4.15 Syringe Head: File Fill

File Fill is a variation of the Fill function which aspirates from a reservoir and dispenses variable volumes across the plate which are inputted from a CSV file identified in the Properties.

Drag and drop the required assets onto the step and check there are no volumetric errors.

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

Property Form Notes
Description Free text  
Source Auto filled from the drag and drop operation, but can be edited from dropdown list  
Destination Auto filled from the drag and drop operation, but can be edited from dropdown list  
Data File Select using the pop up form This is the file defining the dispense volumes.
Preset Select from dropdown list Options for specific tasks

Aspirate

Speed (mm/s) Numeric Aspirate speed of the liquid
Safeguard volume (μL) Numeric Additional volume aspirated into the syringe which remains in the syringe after the dispense.

Pre wet

Use Pre Wet Tick box Aspirate then dispense liquid into the syringe to wet the syringe body and plunger. It is required before the initial aspiration of liquid into the syringe.
Repetitions Numeric Number of aspirate/dispense cycles to perform for the pre-wet.
Speed (mm/s) Numeric The speed the piston rod moves the plunger up during the pre-wet. This will change the speed the liquid is aspirated into the syringe.
Pause (ms) Numeric Time delay between each aspirate-dispense cycle of the pre-wet. It is not applied after the final repetition of the pre-wet.
Shot distance (mm) Numeric The distance the piston rod is forced down by the solenoid for the shot of the pre-wet. Must be less than the up-down distance.
Shot duration (ms) Numeric Duration the solenoid is energised to execute the shot of the pre-wet.
Updown distance (mm) Numeric Distance the plunger moves up/down for the pre-wet.

Pre dispense 1

Use Pre Dispense 1 Tick box Priming dispense(s) performed prior to dispensing to the destination plate.
Volume (μL) Numeric Volume to dispense per repetition of the pre-dispense.
Repetitions Numeric Number of times to dispense the pre-dispense volume.
Suckback threshold (μL) Numeric The maximum dispense volume to which suckback is applied. For solenoid dispenses only.
Pre Dispense pause (ms) Numeric The pause before each pre-dispense repetition. For solenoid dispenses only.
Post Dispense pause (ms) Numeric The pause after each pre-dispense repetition. For solenoid dispenses only.

Pre dispense 2

Use Pre Dispense 2 Tick box Priming dispense(s) performed prior to dispensing to the destination plate.
Volume (μL) Numeric Volume to dispense per repetition of the pre-dispense.
Repetitions Numeric Number of times to dispense the pre-dispense volume.
Suckback threshold (μL) Numeric The maximum dispense volume to which suckback is applied. For solenoid dispenses only.
Pre Dispense pause (ms) Numeric The pause before each pre-dispense repetition. For solenoid dispenses only.
Post Dispense pause (ms) Numeric The pause after each pre-dispense repetition. For solenoid dispenses only.

Dispense

Syringe speed (mm/s) Numeric The speed at which the piston rod moves downwards for solenoid dispenses.
Syringe ramp Select from dropdown list Acceleration profile of the piston rod for a solenoid dispense.
Max Shot Volume (μL) Numeric Maximum volume of a droplet used during a solenoid dispense.
Single Shot Min Volume (μL) Numeric Minimum volume of a droplet used during a solenoid dispense.
Multi Shot Min Volume (μL) Numeric Minimum volume of a droplet used during a multi shot solenoid dispense
Pre Dispense Pause (ms) Numeric The pause before each dispense. For solenoid dispenses only.
Post Dispense Pause (ms) Numeric The pause after each dispense. For solenoid dispenses only.
Syringe Z Offset (mm)  Numeric Z offset when putting syringe into well automatically does an up-down move in between dispenses.
Syringe Offset (mm)  Numeric The distance the syringe is offset from the centre of the well for the dispense.

Direct drive dispense

Move Speed (mm/s) Numeric The speed at which the piston rod moves downwards for direct-drive dispenses.
Inter Dispense pause (ms) Numeric  
Threshold (μL) Numeric The volume above which direct-drive dispensing is enabled.
Auto Finishing Shot  Tick box  This step is optional, though included by default for direct drive dispensing. 
Finishing Shot Volume (µL)  Numeric  

9.4.16 Syringe Head: File Dispense

File Dispense is a variation of the Dispense function which dispenses variable volumes across the plate which are inputted from a CSV file identified in the Properties.  

Drag and drop the required assets onto the step and check there are no volumetric errors. When you have selected the CSV file, a popup appears to show you the dispense pattern from the file data.

A screenshot of a computer screen

Description automatically generated

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

Property Form Notes
Description Free text  
Source Auto filled from the drag and drop operation, but can be edited from dropdown list   
Destination Auto filled from the drag and drop operation, but can be edited from dropdown list   
Data File Select using the pop up form This is the file defining the dispense volumes.
Preset    

 

Dispense

Syringe speed (mm/s) Numeric  The speed at which the piston rod moves downwards for solenoid dispenses.
Syringe ramp Select from dropdown list Acceleration profile of the piston rod for a solenoid dispense. 
Max Shot Volume (µL) Numeric  Maximum volume of a droplet used during a solenoid dispense. 
Single Shot Min Volume (µL)  Numeric  Minimum volume of a droplet used during a solenoid dispense. 
Multi Shot Min Volume (µL)  Numeric  Minimum volume of a droplet used during a multi shot solenoid dispense 
Pre Dispense Pause (ms)  Numeric  The pause before each dispense. For solenoid dispenses only. 
Post Dispense Pause (ms)  Numeric  The pause after each dispense. For solenoid dispenses only. 
Syringe Z Offset (mm)  Numeric  Z offset when putting syringe into well automatically does an up-down move in between dispenses. 
Syringe Offset (mm) Numeric  The distance the syringe is offset from the centre of the well for the dispense. 

 

Direct Drive Dispense

Move Speed (mm/s)  Numeric  The speed at which the piston rod moves downwards for direct-drive dispenses 
Inter Dispense pause (ms)  Numeric   
Threshold (µL)  Numeric  The volume above which direct-drive dispensing is enabled.
Auto Finishing Shot  Tick box This step is optional, though included by default for direct drive dispensing.
Finishing Shot Volume (µL)  Numeric   

 

9.4.17 Syringe Head: Purge

Purge is used to empty remaining reagent from syringes. You must do this before they are removed. 

The pre-set default values should work for most reagents. Use the Advanced properties if you have reagents which are particularly demanding.

Property Form Notes
Description  Free text   
Final Shot Duration (ms)  Numeric  Duration the solenoid is energised.
Source  Auto filled from the drag and drop operation, but can be edited from dropdown list   
Speed (mm/s)  Numeric   

 

9.4.18 Link

If you want to use multiple reagents with one plate, you can perform Aspirate, Dispense, File Fill, File Dispense or Purge steps concurrently.

  1. Create consecutive steps, two or more of the same type. Aspirate or Purge using different reservoirs but fill or dispense into the same plate.
  2. Select (shift +  click) the steps you want to link. When you have done this, the Link function is enabled (on the left)
  3. Click Link and the steps will be linked. Linked steps show a chain-link icon below the text.
    A screenshot of a computer

Description automatically generated

The steps will be performed concurrently not consecutively, speeding up protocol completion.  

To unlink steps, select one or more of the linked steps and the Unlink function (on the right) will be enabled. Click this to unlink.

9.4.19 Labware & Process Modules: Move

Move is a versatile function which you can use to: 

  • reposition assets on the same deck
  • move assets between decks 
  • move assets on and off the shaker and the plate thermal module 
  • create stacks (the same as Place On) 
  • remove assets from stacks (the same as Take Off) 
Property Form Notes
Description  Free text   
Source Item  Auto filled from the drag and drop operation   
Deck Location  Select from dropdown list   

 

Moves are validated so that you do not damage firefly or your labware e.g. you cannot move a tip set to a location on the upper deck as it is too tall. When you select to move an asset, the software shows you a dropdown list of possible destination locations, and which of them are already occupied.

A screenshot of a computer

Description automatically generated

You cannot choose to move an asset to a deck location which is occupied unless the two assets can form a stack e.g. a 96 well plate on a 96 well magnetic block.

A screenshot of a computer

Description automatically generated

If you create an undefined stack, the software will raise an error telling you that you need to download the stack definition. You do not have to do this immediately; you can continue designing but you will not be able to run the protocol without it.

9.4.20 Labware & Processes Modules: Place on

Place on is a very simple function to instruct the grippers to pick up one item and place it on another.

Drag and drop Assets to the top and bottom item positions: if you attempt something which is not possible e.g. making the plate thermal module a top item, the software will not let you drop the item.

Allowed combinations are:

  • Plates can go on the decks, thermal block, magnetic block, and the shaker
  • Magnetic blocks can only go onto the decks
  • Thermal blocks can go onto the decks or the plate thermal module
Property Form
Description Free text
Top item [auto filled from the drag and drop operation, not editable]
Base item [auto filled from the drag and drop operation, not editable]

 

When you use Place on, you create a stack. Check the Errors panel: if you have created an
undefined stack, you will need to download the definition from Community. You do not have to do
this immediately; you can continue designing but you will not be able to run the protocol without
it.

undefined stack error

9.4.21 Labware & Process Modules: Take off

Take off is a very simple function to instruct the grippers to pick up a stacked item and remove it.

Drag and drop an Asset to the bottom item position and firefly software will automatically identify the item directly above it, to be removed. If the stack is plate thermal module - thermal block - plate, if you select the module as the base item, it will remove both the block and the plate as a small stack.

Property Form
Description Free text
Top item [auto filled from the drag and drop operation, not editable]
Base item [auto filled from the drag and drop operation, not editable]

9.4.22 Labware & Process Modules: Heat / Cool

To use the plate thermal module or reservoir thermal module, you must first add it as an Asset.

Use Heat / Cool to control the warming or cooling process. As soon as you start ‘Setup & Loading’, firefly starts heating or cooling the thermal modules as required by the initial Heat / Cool step in the protocol.

Use Properties to set the temperature and behavior:

  • Start and continue’ to have the protocol continue with the next step while the thermal module gets to temperature. If you do this, be aware that depending on your protocol design, the thermal module may be used before your set temperature is reached. You could use a pause to delay using the plate thermal module or aspirating from the reagent reservoirs if temperature is process critical, while enabling other actions e.g. tip loading or user interactions to take place.
  • ‘Start and wait’ means that firefly will do nothing until the temperature setpoint is reached. Heating or cooling rates are dependant on the plate types and reagents used but for example a 96 well plate filled with 50μL water is cooled from 24°C to 4°C in about 5 minutes. 

Use a Move or Place On step to put a thermal block on the plate thermal module so that it is heated or cooled before you use another Move or Place On to put the plate on the thermal block. Alternatively make a stack with the plate and thermal block and place them on at the same time but then the thermal block will be heating or cooling while the plate does.

Plate cooling

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.

Property Form Notes
Description Free text  
Thermal module Select from dropdown list plate thermal module, reservoir thermal module
Behavior Select from dropdown list Start & wait, Start & continue, Stop
Temperature Numeric -20°C to 99°C in steps of 0.1°C

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.

ShakingUse 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.

Property Form Notes
Description Free text  
Behavior Select from dropdown list Start & wait, Start & continue, Stop
Speed Numeric Possible values are 200 - 3000rpm
Duration (hours, minutes, seconds) hours:minutes:seconds Possible values are 1 second - 23 hours, 59 mins, 59 seconds

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.
Property Form Notes
Description Free text Use for the user instructions
Rest state Select from dropdown list List of possible instrument areas to access. Home will return both heads and decks to their home positions.

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.

Property Form Notes
Description Free text This is the text of the note

9.4.26 Pausing

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

Property Form Notes
Description Free text Use to describe purpose of the pause
Duration (hours, minutes, seconds) hours:minutes:seconds
1 second - 23 hours, 59 mins, 59 seconds
Rest State Select from dropdown list
List of possible instrument areas to access. Home will return both heads and decks to their home positions. 

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