Assay development guidelines and protocol notes

This topic provides guidelines and examples on how to vary the protocol settings.

For a description of each setting in the application interface, see Detailed interface description.

Examples of how to vary the Addition step by changing the Incubation and Pause settings

The following examples demonstrate different ways of defining the Incubation Time, Incubation Temp, and Pause After Addition settings to produce different AssayMAP Bravo behaviors.

•	Example 1. Basic addition of a reagent to the samples

•	Example 2: Addition with on-deck incubation

•	Example 3: Addition with a pause

•	Example 4: Addition with on-deck incubation and a pause

•	Example 5: Addition with only Incubation Temp Set and Pause Activated

Example 1. Basic addition of a reagent to the samples

This example shows the settings for a simple addition step that uses the bare AssayMAP probes to transfer 10 µL from the Reagent plate at deck location 5 into the Sample plate at deck location 4.

Figure. Example 1 settings

 

Table Example 1: AssayMAP Bravo actions during the protocol

 

Action	Deck location
a Uses the bare probes to aspirate 10 µL from the Reagent plate into the syringes.	5
b Dispenses the 10 µL into the Sample plate.	4
c Performs three mix cycles in the Sample plate to mix the contents.	4
d Performs three syringe washes at the wash station.	1
e Continues on to Addition Steps 2 to 4.	varies

When to use these settings: In general, set the Incubation Time (min) to 0 and the Incubation Temp (°C) to OFF for addition steps where incubation and deck manipulation are not required.

However, if an Addition step with no incubation follows a step that includes incubation, make sure that you account for the ramp time to change the temperature. For details, see Additional incubation control considerations.

 

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Example 2: Addition with on-deck incubation

This example uses the Peltier Thermal Station to automate an incubation step. The addition step settings include on-deck incubation after the liquid transfer to the Sample plate. The on-deck incubation of the Sample plate is activated by setting the Incubation Time (45 min) and the Incubation Temp (37 °C).

Figure. Example 2 settings

 

Table Example 2: AssayMAP Bravo actions during the protocol

 

Action	Deck location
a Sets the Peltier Thermal Station to 37 °C. Note: The heater is activated before the liquid transfer because it takes about 5 minutes for the temperature to reach the set point.	4
b Uses the bare probes to aspirate 10 µL from the Reagent plate into the syringes.	5
c Dispenses the 10 µL into the Sample plate.	4
d Performs three mix cycles in the Sample plate to mix the contents.	4
e Starts the incubation timer when the Peltier Thermal Station reaches 37 °C.	–
f Performs three syringe washes at the wash station.	1
g Remains idle for the remainder of the 45-minute incubation time.	–
h Continues on to Addition Steps 2 to 4.	varies

When to use these settings: Set the Incubation Time and Incubation Temp values for all addition steps where the Sample plate incubation will be conducted automatically on the AssayMAP Bravo deck.

 

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Example 3: Addition with a pause

This example shows settings for an addition step that includes a pause after the liquid transfer to the Sample plate.

Figure. Example 3 settings

 

Table Example 3: AssayMAP Bravo actions during the protocol

 

Action	Deck location
a Uses the bare probes to aspirate 10 µL from the Reagent plate into the syringes.	5
b Dispenses the 10 µL into the Sample plate.	4
c Performs three mix cycles in the Sample plate to mix the contents.	4
d Performs three syringe washes at the wash station.	1
e A Pause message appears on the screen and the AssayMAP Bravo Platform stops executing new tasks. The action remains paused until you click the Continue button that appears on the screen.	–
f Continues on to Addition Steps 2 to 4.	varies

When to use these settings: Select the Pause After Addition option to pause the run so that you can do the following:

•	Replace used pipette tips with new pipette tips.

•	Remove the Sample plate from the deck for off-deck incubation.

 

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Example 4: Addition with on-deck incubation and a pause

This example uses the Peltier Thermal Station to automate an incubation step. After the incubation, the run will pause to allow the operator to modify something on the deck. The on-deck incubation of the Sample plate is activated by setting the Incubation Time (60 min) and the Incubation Temp (60 °C). The Pause After Addition check box is also selected.

Figure. Example 4 settings

 

Table Example 4: AssayMAP Bravo actions during the protocol

 

Action	Deck location
a Sets the Peltier Thermal Station to 60 °C. Note: The heater is activated before the liquid transfer because it takes about 5 minutes for the temperature to reach the set point.	4
b Uses the bare probes to aspirate 10 µL from the Reagent plate into the syringes.	5
c Dispenses the 10 µL into the Sample plate.	4
d Performs three mix cycles in the Sample plate to mix the contents.	4
e Starts the incubation timer when the Peltier Thermal Station reaches 60 °C.	–
f Performs three syringe washes at the wash station.	1
g Remains idle for the remainder of the 60-minute incubation time.	–
h A Pause message appears on the screen and the AssayMAP Bravo Platform stops executing new tasks. The action remains paused until you click the Continue button that appears on the screen. Note: The Peltier Thermal Station remains at temperature (60 °C in this case) until the Pause message is cleared.	–
i Continues on to Addition Steps 2 to 4.	varies

When to use these settings: Use these settings to incubate a plate.

 

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Example 5: Addition with only Incubation Temp Set and Pause Activated

This example uses the Peltier Thermal Station to partially automate an incubation step. The on-deck incubation of the Sample plate is activated by setting the Incubation Temp (60 °C). However, no Incubation Time is specified. The Pause After Addition option is selected, which will cause the incubator to be active during the pause so that the operator can control the duration of the incubation.

Figure. Example 5 settings

 

Table Example 5: AssayMAP Bravo actions during the protocol

 

Action	Deck location
a Sets the Peltier Thermal Station to 60 °C. Note: The heater is activated before the liquid transfer because it takes about 5 minutes for the temperature to reach the set point.	4
b Uses the bare probes to aspirate 10 µL from the Reagent plate into the syringes.	5
c Dispenses the 10 µL into the Sample plate.	4
d Performs three mix cycles in the Sample plate to mix the contents.	4
e Performs three syringe washes at the wash station.	1
f A Pause message appears on the screen and the AssayMAP Bravo Platform stops executing new tasks. The action remains paused until you click the Continue button that appears on the screen. Note: The Peltier Thermal Station remains at temperature (60 °C in this case) until the Pause message is cleared.	–
g Continues on to Addition Steps 2 to 4.	varies

When to use these settings: Use this combination of settings when it is necessary to apply a robust seal to the plate, but still use the on-deck incubator to heat or cool the samples.

Note: In this case, the operator is responsible for timing the incubation step because the system does not keep track of the time that the plate was removed or returned to the deck.

 

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Additional incubation control considerations

 

The Peltier Thermal Station requires time to reach the temperature set point. Therefore, the device must be activated before incubation at the set temperature begins.

 

The time that it takes the Peltier Thermal Station to reach the set temperature is called “ramp” time. The temperature is in flux during the ramp time. The duration the ramp time greatly depends on whether the Peltier Thermal Station is being used to actively control the temperature to the next set point or not. The following table provides approximate temperature ramp times for common incubation scenarios.

Table Examples of approximate ramp time (min) to reach target temperature

 

Scenario	Uses Peltier Thermal Station?	Start temp (°C)	Target temp (°C)	Ramp time (min), approximate
1	Yes	25	60	< 5
2	Yes	25	10	< 5
3	Yes	60	10	< 10
4	Yes	37	25	< 5
5	No	37	25	> 15
6	No	60	25	> 30
7	No	10	25	> 20

The following examples illustrate why temperature ramp times are an important consideration when defining an In-Solution Digestion method.

Example 1: Addition with incubation followed by addition with heater turned off

Figure. Example 1 settings

 

In this example:

•	Addition 1 requires an incubation of 45 minutes at 60 °C.

•	Addition 2 does not require incubation, so the Incubation Temp is set to OFF.

Intuitively, it makes sense to turn off the incubator if it is not required, but this could have unintended consequences for Addition 2 and subsequent additions. After completing Addition 1, the Peltier Thermal Station will be turned off in preparation for Addition 2, but its temperature will still be very close to 60 °C. According to the preceding table of example ramp times, the Peltier Thermal Station requires at least 30 minutes to return to ambient temperature (~25 °C). The elevated temperature at deck location 4, where the Peltier Thermal Station is installed, could be problematic if later addition steps are sensitive to elevated temperature. In most cases, this problem can be mitigated by using the Peltier Thermal Station to actively cool the temperature back to ambient temperature, as described in Example 2.

 

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Example 2: Addition with incubation followed by addition with heater at ambient temp

Figure. Example 2 settings

 

In this example:

•	Addition 1 requires an incubation of 45 minutes at 60 °C.

•	Addition 2 requires at 25 °C (ambient temperature) at deck location 4.

These settings will cause the Peltier Thermal Station to actively cool the temperature from Addition 1 to ambient temperature for Addition 2. The approximate time required is 5 minutes for this temperature change. See the preceding table of example ramp times.

 

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Error messages

Before executing an In-Solution Digestion: Single Plate method, the application performs several error checks to ensure that a feasible run has been specified. For a description of the types error checks, see Control panel.

The error checks are performed when the operator presses the Run Digestion button. If a problem is identified, an error message will display in the Method Status box of the form before any run tasks are executed.

The following table lists the error messages, reasons for the error, and a description of how to correct the error.

 

Method Status error message	Reason for error	How to correct the error
File chosen is not a valid Method file.	The method file selected is for a different application.	Click the Browse for a Method button, and then select a method file from the following folder: …\VWorks Workspace\Methods\AM In Solution Digestion Single Plate v1.0
File type is incorrect. Please choose a correct Method file.	The file selected is not a method file.	Click the Browse for a Method button, and then select a method file from the following folder: …\VWorks Workspace\Methods\AM In Solution Digestion Single Plate v1.0
No file chosen. Please select a file from the file browser and load values again.	The Load Method button was pressed before a method file was selected.	Click the Browse for a Method button, and then select a method file from the following folder: …\VWorks Workspace\Methods\AM In Solution Digestion Single Plate v1.0 Click Load Values to load the method.
Error: Total volume will exceed Sample Labware capacity (0 µl). Please select a larger capacity Sample Labware.	No labware is selected for the Sample Plate.	Under Input Sample Settings, select the labware that you are using from the Sample Plate Labware list.
Error: Total volume will exceed Sample Labware capacity (n µl). Please select a larger capacity Sample Labware.	The initial sample volume exceeds the capacity of the selected labware type.	Do one of the following: • Select a labware type with a larger capacity from the Sample Plate Labware list. • Reduce the value in the Starting Sample Volume box to a number that is within the capacity of the current labware selection. For a list of maximum volumes by labware type, see Labware.
Error: Sample Plate is a type of deep well labware. Tips are needed in all steps.	Some of the addition steps specify the use of the AssayMAP bare probes, which cannot access the bottom of most deep-well plates. Pipette tips are required for addition steps that include a deep-well plate on the AssayMAP Bravo Platform.	Do one of the following: • Select the Use Tips for Addition check box for each Addition step to ensure that pipette tips are used for the transfers instead of the AssayMAP probes. • If you do not want to use pipette tips for the transfers, ensure that no deep-well plates are selected in the Sample Plate Labware list and the Labware Selection lists for the Addition steps.
Error: Check Step 1 for volume/labware/tip combination.	The Volume input for one or more of the Addition Steps is greater than the volume capacity for the selected labware type.	Do one of the following for the corresponding Addition step: • Select a labware type with a larger capacity from the Labware Selection list. • Reduce the value in the Addition Volume box to a number that is within the capacity of the current labware selection. For a list of maximum volumes by labware type, see Labware.
Error: Check Step 1 for volume/labware/tip combination. Error: Total volume will exceed Sample Labware capacity. Please select a larger capacity Sample Labware.	The cumulative volume in the sample plate, after all addition steps have completed, would be greater than the capacity of the selected Sample plate labware type.	Do one of the following: • Select a labware type with a larger capacity from the Sample Plate Labware list. • Reduce the values in the Starting Sample Volume box and/or the Addition Volume boxes to volumes that are within the capacity of the current labware selection. For a list of maximum volumes by labware type, see Labware.
There are no reagent steps included on the form. Please double check and start the protocol again.	The Addition Name for all four addition steps is set to None, which causes all steps to be skipped.	For each addition step you want to include, select or type a name in the Addition Name box. Ensure that you have entered settings for at least one addition step in the Input Addition Step Settings area.
Error: Incubation temperature for Addition step 1 is out of range.	The Incubation Temp (°C) is set to a value that is outside of the allowable range (4 to 70 °C).	If you want to specify an on-deck incubation, enter a value within the allowable range (4 to 75 °C) in the Incubation Temp (°C) box. If you do not want to specify a temperature, select OFF. IMPORTANT For guidelines on how to control the temperature changes between addition steps, see Additional incubation control considerations.
Error: No labware associated with Addition step 1.	The Labware Selection is set to None for one or more addition steps.	For each addition step you want to include, select the corresponding labware from the Labware Selection list.
Error Check Step 1 for volume/labware/tip combination.	A deep-well plate is selected for one or more of the addition steps, but these steps are specified to use AssayMAP Probes, which cannot access the bottom of most deep-well plates.	Do one of the following: • Select the Use Tips for Addition check box for each Addition step to ensure that pipette tips are used for the transfers instead of the AssayMAP probes. • If you do not want to use pipette tips for the transfers, ensure that no deep-well plates are selected in the Labware Selection lists for the Addition steps.
Error: Addition step 1 labware is a reservoir, lids cannot be used.	The selected labware type cannot have a lid, but the Plate Lidded option is also selected.	Do one of the following: • Clear the Plate Lidded check box for this Addition step. • Select a type of labware that can use a lid in the Labware Selection list for this Addition.
Error in the new process name. Please enter a valid name.	No file name was specified when attempting to save a method file.	In the Save Method area, type a Name. If applicable, select the Overwrite if Name Exists check box, and then click Save.

 

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Automation movements during the protocol

The automation movements are dependent on the choices made during the method definition. See the Detailed interface description.

 

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