Running the protocol

The In-Solution Digestion: Single Plate protocol performs up to four successive liquid-addition steps. Each addition step transfers a reagent or mixture of reagents into a common sample plate, followed by incubation on or off deck. The sample volume, incubation time, incubation temperature, labware, and other parameters can be customized for each of the four addition steps, for example:

•	Denaturant mixture is added to the samples.

•	Samples are denatured and reduced at elevated temperature.

•	The samples are alkylated.

•	The samples are diluted with Dilution mixture.

•	Protease is added to the samples.

Before you start

Ensure that you:

•	Prepare the reagents. See Preparing the reagents.

•	Prepare the samples. See Preparing the samples.

•	Runs that require pipette tips. Prepare the 96AM Cartridge & Tip Seating Station. For details, see Preparing the 96AM Cartridge & Tip Seating Station.

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

Setting up the protocol

Before you start the protocol, make sure the appropriate selections and values are specified in the In-Solution Digestion application.

To set up the In-Solution Digestion protocol:

1	If not already open, open the Protein Sample Prep Workbench App Library. See Accessing an application.

2	Locate In-Solution Digestion: Single Plate, and then click App.

 

The In-Solution Digestion: Single Plate application opens.

3	Optional. Click Restore Defaults to set all Application Settings to their defaults.

4	In the A: Select Method area, click , select the method, and then click Load Values to display all the settings and the Deck Layout associated with the selected method.

Note: By default, the methods are stored in the C:\VWorks Workspace\Methods\AM In Solution Digestion Single Plate v1.0 folder.

To run a saved method exactly as written, skip to step 7. Otherwise, proceed to step 5 to make changes.

5	Under B. Input Sample Settings, verify the settings, or make any required changes to meet the needs of your run. For more details, see B: Input Sample Settings.

6	Under C. Input Addition Step Settings, verify the settings, or make any required changes to meet the needs of your run. For more details, see C: Input Addition Step Settings.

7	Under D. Update Deck Layout, click Update Deck Layout to refresh the deck image so that it reflects the selections for the current method, including any changes that you have made.

 

Incorrect labware selections can cause a hardware collision, resulting in equipment damage. Ensure that the labware selected in the method matches the physical labware present on the Bravo deck.

 

8	To save a new or edited method, specify the method Name in the Save Method area, select Overwrite if Name Exists if applicable, and then click Save.

For more details, see Control panel.

 

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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 and source bottles.

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 decrease the volumes.

If your In-Solution Digestion protocol requires pipette tips, you must use pipette tips for the mock run. For instructions on how to set up the pipette tips in the 96AM Cartridge & Tip Seating Station, see Preparing the 96AM Cartridge & Tip Seating Station.

 

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Starting the protocol run

 

The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

 

 

Incorrect labware selections can cause a hardware collision, resulting in equipment damage. Ensure that the labware selected in the method matches the physical labware present on the Bravo deck.

 

To start the protocol run:

1	Place the items at the assigned deck locations, as shown in the Deck Layout of the application interface:

•	Place the filled sample plate at deck location 4, and place the filled reagent plates at their respective deck locations.

•	Verify that all plates that require lids have been properly lidded, and no lids are present on other labware.

•	If pipette tips are required for the run, place the prepared 96AM Cartridge & Tip Seating Station at deck location 2. For details, see Preparing the 96AM Cartridge & Tip Seating Station.

2	Ensure that all labware are properly seated on the deck.

 

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 to start the run.

To monitor the progress of the run, check the Status box.

 

To stop a run in an emergency, use the hardware Emergency Stop button.

 

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.

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.

 

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Cleaning up

When the protocol run is finished, make sure you:

•	Remove all labware from the AssayMAP Bravo deck.

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

•	Run the System Shutdown utility. See System Startup/Shutdown v2.2.

 

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.

 

 

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