Running the protocol

The On-Cartridge Reaction protocol does the following:

•	Washes the syringes.

•	Equilibrates the cartridges.

•	Loads the Reaction reagent onto the cartridges and incubates the reaction at the temperature set point.

•	Collects soluble reaction products.

•	Removes non-target elements from the immobilized reaction product.

•	Elutes the immobilized reaction product from the cartridges.

For some of these operations the cartridges are mounted on the syringe probes, while for other operations the cartridges are parked in the Cartridge & Tip Seating Station.

Before you start

Ensure that you:

•	Prepare the reagents. See Preparing the reagents.

•	Run the Startup protocol to prepare the AssayMAP Bravo Platform for the run. See System Startup/Shutdown v2.2.

•	If you have not already done so, transfer the prepared cartridges to the Cartridge & Tip Seating Station. See Cartridge Transfer v1.1.

This application uses cartridges that have been prepared during a preceding AssayMAP application, typically, Affinity Purification.

 

Do not allow cartridges with immobilized substrate to dry out. If the cartridges do dry out, do not use them.

 

Setting up the protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the On-Cartridge Reaction application.

To set up the protocol:

1	Open the App Library. See Accessing an application.

2	Locate On-Cartridge Reaction, and then click App.

 

The On-Cartridge Reaction application opens.

 

3	Do one of the following:

•	To use the current settings that appear in the form, go to Starting the protocol run.

To restore the default settings, click Restore Defaults.

•	To use a saved method, under Select Method, click , and locate and select the method file. Click Load to display all the settings associated with the selected method.

 

To use a saved method exactly as written, go to Starting the protocol run.

To modify the settings, proceed to step 4.

4	In the Application Settings area, specify the Number of Full Columns of Cartridges to use during the run. The position of the columns of cartridge in the tip seating station must match the positions of the samples and solutions in the plates on the deck.

Range: 1–12

Default: 1

 

If the column selection is greater than the actual number of columns used, the Bravo Platform will apply too much force when mounting the cartridges, which can cause damage to both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 10 columns, but only 1 column of cartridges are in the seating station, the head will apply 10 times more force than what is required. To prevent potential equipment damage, ensure that the column selection is correct.

 

 

If the column selection in the software is less than the actual number of cartridges used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 10 columns of cartridges are in the seating station, the head will apply 1/10th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable. To obtain expected instrument performance, ensure that the column selection is correct.

 

 

Each full column must contain eight cartridges. If a column contains fewer than eight packed cartridges, use the AssayMAP Resin-Free cartridges to fill the empty column positions.

 

5	In the Application Settings table, select the check boxes of the steps that you want to perform and enter values for the selected steps. For details, see Application Settings table.

6	In the Labware Table area, select the labware you are using for the protocol run.

7	To save the method, specify the method Name in the Save Method area. If applicable, select Overwrite if Name Exists, and then click Save to save the settings in a method file.

 

 

Return to top

Application Settings table

The following table gives a brief description of each setting. For more details, including the practical and full ranges of values for a given setting, see the Assay development guidelines and protocol notes.

 

Step	Description
Initial Syringe Wash	Conducts internal syringe washes on all 96 of the syringes, using the wash station, to remove potential contaminants. Select the Initial Syringe Wash step and specify the number of wash cycles. Clear the check box if you are certain that the syringes are clean. This step is selected by default, and is conducted at the very beginning of the run. Wash Cycles: Default: 3
Equilibrate	Ensures that the resin bed has been fully equilibrated using buffer conditions that are appropriate for the reaction step. The Equilibrate step aspirates the Equilibration Buffer into the syringes, picks up the cartridges, and then dispenses a specified volume through the cartridges at the specified flow rate (µL/min). Specify the volume, the flow rate, and the number of syringe wash cycles to be performed following equilibration. This step is selected by default. Volume: Default: 50 Note: Setting the volume to zero will skip all tasks associated with the Equilibrate step except syringe washing at the wash station. Flow Rate: Default: 10 Wash Cycles: Default: 1
Collect Flow Through	Dispenses the flow-through during the Equilibrate step into Flow Through Collection (deck location 7) if selected. Since the cartridges have immobilized substrate, this option allows you to determine if any substrate is coming off the cartridges during this step. If not selected, discards the equilibration flow-through at the wash station. This step is not selected by default.
Reaction	Aspirates the Reagent through the cartridges in two steps, and then aspirates a chase volume: 1 Initial Aspiration. Aspirates 4 µL of liquid at a flow rate of 10 µL/min (minimum volume). 2 Secondary Aspiration. Aspirates any additional volume at a flow rate appropriate to satisfy the Duration setting. For example, if the volume is set at 6 µL and the time was set for 30 minutes, the remaining 2 µL of the 6 µL volume would be aspirated at a flow rate of ~0.068 µL/min (2 µL / (30 min – 0.4 min)). 3 Reaction Chase Aspiration. Aspirates the chase at the specified volume and flow rate through the cartridges. The Reaction flow-through and chase are collected in the syringes, and then dispensed into the Flow Through Collection plate (deck location 7), unless Combine with Eluate is selected. In which case, the Reaction flow-through and chase are dispensed into the Eluate Collection plate (deck location 9). This step is selected by default. Volume: Default: 6 Wash Cycles: Default: 1
Temperature	Specifies the set point temperature of the Peltier Thermal Station at deck location 4 during the Reaction step. The actual temperature in the cartridges will be less than this setting. For example, a cartridge bed temperature of approximately 37 °C is obtained with a setting of 45 °C when using the Red PCR Plate Insert and a PCR plate. This temperature differential is due to the heat loss that occurs as the heat transfers across the thermal plate insert, the air gap between the plate insert and the wells of the plate, the plastic of the plate, the solution in the well, and the cartridge resin bed. IMPORTANT The temperature affects the amount of excess volume that must be in the well at the beginning of the run. A higher temperature will cause more evaporation and, therefore, require more excess volume. See Determining the volume overage for deck location 4. Temperature °C: Default: 25
Duration	Specifies the total length of time to aspirate the Reagent (deck location 4) through the cartridges, including the initial and secondary aspiration steps. Note: This setting governs only the aspiration of Reagent through the cartridges. IMPORTANT The duration affects the amount of excess volume that must be in the well at the beginning of the run. Longer durations will cause more evaporation and, therefore, require more excess volume. See Determining the volume overage for deck location 4. Time (minutes): Default: 30
Reaction Chase	If selected, aspirates the Chase Buffer (deck location 3) through the cartridges to flush soluble reaction products out of the cartridge bed and into the syringes. This step occurs immediately after the aspiration of Reagent, combining the flow-through of the initial and secondary aspiration with the Chase Buffer within the syringes. This combination is collected at the Flow Through Collection plate (deck location 7) unless Combine With Eluate is selected. In which case, the combined solution is collected at the Eluate Collection (deck location 9). Volume: Default: 25 Flow Rate: Default: 5
Combine with Eluate	Specifies where the Reaction initial and secondary aspiration and Reaction Chase will be collected: • If selected, collects the initial and secondary aspiration and Reaction Chase at Eluate Collection (deck location 9). • If not selected, collects the initial and secondary aspiration and Reaction Chase at Flow Through Collection (deck location 7). The step is not selected by default.
Cup Wash 1	Washes the cups of the parked cartridges using Cartridge Wash Buffer 1, and then performs a syringe wash at the wash station. Specify the buffer volume for the cup wash and the number of wash cycles. This step is selected by default. Volume: Default: 25 Wash Cycles: Default: 2 Note: Each wash cycle consists of a cup wash followed by syringe wash.
Internal Cartridge Wash 1	Aspirates the Cartridge Wash Buffer 1 (deck location 5) into the syringes, and then dispenses it through the cartridges into the wash station (deck location 1), unless Collect Flow Through is selected, which dispenses the flow-through to Flow Through Collection (deck location 7). This step is selected by default. Volume: Default: 50 Flow Rate: Default: 20 Wash Cycles: Default: 1
Collect Flow Through	Dispenses the flow-through during the Internal Cartridge Wash 1 step into the Flow Through Collection plate (deck location 7). • Select the option if you want to collect the flow-through from Internal Cartridge Wash 1 for downstream processing or analysis. • Clear the check box if you want to dispense the flow-through from Internal Cartridge Wash 1 into the wash station. This step is not selected by default.
Cup Wash 2	Washes the cups of the parked cartridges using Cartridge Wash Buffer 2, and then performs a syringe wash at the wash station. Specify the buffer volume for the cup wash and the number of wash cycles. This step is selected by default. Volume: Default: 25 Wash Cycles: Default: 2 Note: Each wash cycle consists of a cup wash followed by syringe wash.
Internal Cartridge Wash 2	Aspirates the Cartridge Wash Buffer 2 into the syringes, and then dispenses it through the cartridges into the wash station, unless Collect Flow Through is selected, which dispenses the flow-through to Flow Through Collection (deck location 7). This step is selected by default. Volume: Default: 50 Flow Rate: Default: 20 Wash Cycles: Default: 1
Collect Flow Through	Dispenses the flow-through during the Internal Cartridge Wash 2 step into the Flow Through Collection plate (deck location 7). • Select this check box if you want to collect the flow-through from Internal Cartridge Wash 2 for downstream processing or analysis. • Clear the check box if you want to dispense the flow-through from Internal Cartridge Wash 2 into the wash station. This step is not selected by default.
Stringent Syringe Wash	Washes the syringes with the Elution Buffer followed by a syringe wash at the wash station. Select this step when rigorous cleaning of the syringes is necessary to prevent carryover. The step is selected by default. This step is selected by default. Volume: Default: 50 Note: Setting the volume to zero will skip all tasks associated with the Stringent Syringe Wash step. Wash Cycles: Default: 2
Elute	Elutes the immobilized reaction product from the cartridges using the Elution Buffer, and then performs syringe washes at the wash station. Always select this step to remove the immobilized reaction product from cartridges in a normal run. The step is selected by default. This step is selected by default. Volume: Default: 25 Note: Setting the volume to zero will skip all tasks associated with the Elute step except syringe washing at the wash station. Flow Rate: Default: 5 Wash Cycles: Default: 1
Eluate Discard	Discards the first specified volume of eluate at the flow rate specified for the Elute step. The volume can equal, but not exceed the Elute volume. Volume: Default: 0 This step is not selected by default.
Existing Collection Volume	Specifies the volume of liquid present in the Eluate Collection plate (deck location 9) at the start of the run. Volume: Default: 0
Final Syringe Wash	Conducts the specified number of internal syringe washes at the wash station. Select this step to clean the syringes thoroughly before running a different assay. This step is selected by default. Wash Cycles: Default: 3

Return to top

About performing a mock run (optional)

If you are unfamiliar with the protocol and would like to see how it operates before running it with valuable samples and reagents, you can perform a mock run. A mock run uses empty or water-filled labware.

You prepare for a mock run the same way you would prepare for a real protocol run, except that you use empty labware for a totally dry run or labware containing water for a wet run. To decrease the run time, you can increase the flow rates to 500 µL/min and decrease the volumes. Use the AssayMAP Resin-Free cartridges instead of packed cartridges for mock runs.

 

The protocol will not run if cartridges are missing.

 

 

Return to top

Starting the protocol run

To start the protocol run:

1	Review the selections and value specifications in the protocol to confirm they are correct. Make sure:

•	The physical layout on the Bravo deck matches the graphic shown in the Deck Layout area.

•	In the Labware Table area, the selections reflect the labware you are using for the protocol run.

2	Make sure the labware are properly seated on the Bravo deck.

 

Use only the labware specified, and place them at the AssayMAP Bravo deck locations specified in the form. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

 

 

Improperly seated labware can cause a hardware collision, resulting in equipment damage. Ensure that all labware are properly seated within the alignment features of their respective platepads.

 

3	Click Run Protocol.

An error message appears if the software detects an error in the form settings, for example, if an entered value is outside the limits set for a field. If this happens, follow the directions in the message to select Pause and Diagnose, and then select Abort process. After setting the appropriate values, click Run Protocol to restart the run.

Note: For the range of acceptable values for a given setting, see the Assay development guidelines and protocol notes.

To monitor the progress of the run, check the Status box in the upper right corner of the form.

 

After the protocol run starts, you can walk away from the AssayMAP Bravo Platform for the duration of the protocol, which is indicated in the Status box.

 

To stop a run in an emergency, use the emergency stop pendant.

 

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses.

 

Protocols should not be paused to change the settings. However, if you need to change a setting in a running protocol, click Pause, and then click Abort process in the dialog box that appears. Change the setting, and then clear the check boxes of the steps in the On-Cartridge Reaction application that have been completed successfully. Restart the protocol to complete the remaining steps.

 

To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

 

Return to top

Cleaning up

When the protocol run is finished, make sure you:

•	Remove all labware from the Bravo deck.

•	Discard solutions, organic waste, and used labware following appropriate waste disposal procedures.

 

Make sure you discard the chemical waste and used labware according to your lab’s waste disposal procedures and in compliance with all local, state, and federal safety regulations.

 

 

Return to top