Reagent Transfer v2.1

The Reagent Transfer v2.1 utility transfers samples or reagents from a Source Plate to a Destination Plate. This utility is useful for a wide variety of sample manipulation and reagent preparation operations. The Reagent Transfer v2.1 features include the ability to:

•	Save and reuse transfer methods.

•	Conduct liquid transfers using either the bare probes of the AssayMAP head or 250-µL pipette tips.

•	Control the liquid-handling processes, including:

–	Use reverse pipetting or forward pipetting modes, which can increase pipetting accuracy depending on the liquid properties and volume being transferred.

–	Select from a list of liquid classes or enter a custom liquid class.

–	Specify whether to conduct syringe drying cycles and pre-wet cycles.

•	Control the locations of the liquid transfer in both the Source and Destination plates.

Before you start

Prepare the Source Plate and Destination Plate with their intended reagents. Ensure the Source Plate has the required volume overage for the labware type. For details, see the Labware requirements table.

Labware requirements

The following table lists the labware options for Source Plate and Destination Plate at Bravo deck locations 7 and 8.

 

 

Labware options for deck locations 7 and 8	Manufacturer part number*	Source Plate volume overage (well)**
96 Eppendorf 30129300, PCR, Full Skirt, PolyPro	Eppendorf 30129300	10 µL
96 Bio-Rad PCR, Hard-Shell, Low-Profile, Full Skirt	Bio-Rad HSP‑9611	10 µL
96 Greiner 652270, PCR, Full Skirt, PolyPro	Greiner 652270	10 µL
96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom	ABgene AB-1127	30 µL
96 Greiner 650201_U-Bottom, Clear PolyPro	Greiner 650201	20 µL
96 Greiner 650207_U-Bottom, White PolyPro	Greiner 650207	20 µL
96 Greiner 651201_V-Bottom, Clear PolyPro	Greiner 651201	20 µL
96 Costar 3363, PP Conical Bottom	Corning Costar 3363	20 µL
96 Greiner 675801, Half Area, Flat-Bottom, UV Star	Greiner 675801	25 µL
12 Column, Low Profile Reservoir, Natural PP	Agilent 201280-100	3 mL
8 Row, Low Profile Reservoir, Natural PP	Agilent 201282-100	4.5 mL
Reservoir, Seahorse 201254-100, PP, no walls, pyramid bottom	Agilent 201254-100	20 mL
Reservoir, Axygen Scientific RES-SW96-LP, 86mL pyramid bottom	Axygen Scientific RES‑SW96‑LP	20 mL
96 V11 Manual Fill Reservoir	Agilent G5498B#049	35 mL
96AM Receiver Plate	Included with Agilent cartridge rack	Not applicable
96 Eppendorf 96/500 V-bottom, Clear, PolyPro***	Eppendorf 96/500	25 µL
96 Eppendorf 96/1000 U-bottom, Clear, PolyPro***	Eppendorf 96/1000	25 µL
96 Waters 186005837, Clear PolyPro***	Waters 186005837	30 µL
*For dimensionally equivalent alternatives, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. **This overage does not include the volume required for reverse pipetting. The volume is listed by well, where a well is defined as an individual well in a 96-well plate. But in labware where multiple syringes can draw from a common source, a well is defined as that common source. For example, a 12-Column Low-Profile Reservoir has 12 wells, an 8-Row Low-Profile PolyPro Reservoir has 8 wells, and a Seahorse 201254-100, PP, No Walls, Pyramid-bottom Reservoir has only 1 well. ***Pipette tips are required for reagent transfers using these deep-well plate options.

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Accessing the Reagent Transfer utility

To access the Reagent Transfer utility:

In the Utility Library, locate the Reagent Transfer utility, and then click Utility.

 

The following figure shows the Reagent Transfer v2.1 utility form.

 

 

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Setting up the method

To set up the Reagent Transfer utility method:

1	Do one of the following:

•	To create a new method, proceed to step 2.

•	To use or modify an existing method:

–	Under Select Method, click the Browse for a Method button . Locate and select the method file.

Note: Typically, the methods are stored in the following folder: C:\VWorks Workspace\Methods\AM Reagent Transfer v2.1.

–	Click Load to display all the settings associated with the selected method.

–	To use a saved method exactly as written, skip to Running the Reagent Transfer utility. To modify the method, proceed to step 2 to make changes.

 

2	Under Application Settings area, specify the following:

a	Pipette Tip Settings

 

Property	Description
Use Pipette Tips	The option to use 250 µL pipette tips to transfer the liquid: • To use pipette tips, select the check box. • To use the bare probes on the AssayMAP head, clear the check box. Note: Ensure that the labware selection is appropriate for the bare probes. For details, see the Labware requirements table. Default: Not selected
Transfer Pipette Tips from Deck Location 6	If the Use Pipette Tips check box is selected, specify whether to transfer pipette tips using the Reagent Transfer utility. CAUTION A collision can occur if any pipette tips are present in the source tip box outside of the range specified on the form. • To transfer the pipette tips from a source tip box at deck location 6 to the empty 96AM Cartridge & Tip Seating Station at deck location 2, select the check box. IMPORTANT The Pipette Tip Box must contain contiguous columns of 8 pipette tips. • To use pipette tips that are already present in the 96AM Cartridge & Tip Seating Station at deck location 2, clear the check box. For example, if you do a run and transfer the pipette tips from deck location 2 to the 96AM Cartridge & Tip Seating Station, and then want to do additional runs with the same setting, the pipette tips can be reused. IMPORTANT The pipette tips must be in contiguous columns of 8 pipette tips. Default: Not selected
Columns of Pipette Tips at Deck Location 6	If the Transfer Pipette Tips from Deck Location 6 check box is selected, specify the range of full columns of pipette tips present in the source tip box (deck location 6) at the beginning of the run. Default: 1 to 12 Range: • Starting column: 1–12 • Ending column: 1–12

b	Source Plate Settings

 

Property	Description
Columns of Samples to Transfer	The range of wells (full columns) in the Source Plate (deck location 7) to be used for the reagent transfer. Note: If using bare probes, use the default range of 1 to 12. When using bare probes, the head moves as if it is moving all 96 positions from the Source Plate to the Destination Plate. Therefore, liquid will be moved from every position on the Source Plate where it exists to every corresponding position on the Destination Plate. Default: 1 to 12 Range: • Starting column: 1–12 • Ending column: 1–12
Initial Well Volume	The volume of liquid present in the wells of the Source Plate (deck location 7) before transferring the liquid. For labware where multiple syringes draw from a common source, a “well” is defined as that common source. For example, a 12-Column Low-Profile Reservoir has 12 wells, an 8-Row Low-Profile PolyPro Reservoir has 8 wells, and a Seahorse 201254-100, PP, No Walls, Pyramid-bottom Reservoir has only 1 well. For these labware types, the Initial Well Volume is calculated as: For example, in the Seahorse 201254-100, PP, No Walls, Pyramid-bottom Reservoir the overage required is 20 mL plus 96 times the volume to be transferred (e.g., 100 µL) divided by 96: This value is used to calculate the appropriate height for the pipette tips or probes during the aspiration process, and to facilitate proper dynamic-tip behavior. Note: Using a value of 0 will disable dynamic-tip movement and force the probes or tips to aspirate from the minimum distance from the well bottom of the selected labware. This might result in the pipette tip or probe exterior being exposed to liquid that might not be removed at the wash station. Default: 0 (µL) Range: 0–1500 (µL)
Volume to Transfer	The volume of liquid per syringe to transfer from the Source Plate (deck location 7) to the Destination Plate (deck location 8). If this volume setting is greater than the capacity of the probes or pipette tips, the transfer will be evenly split into multiple aspirate-and-dispense cycles. Default: 10 (µL) Range: 0–1200 (µL) Note: The Automatic Liquid Class setting is designed for a 5 µL minimum. A volume less than 5 µL might require a custom liquid class.

c	Destination Plate Settings

 

Property	Description
Columns to Receive Samples	The range of full columns in the Destination Plate (deck location 8) to which the samples will be transferred. When using pipette tips, this range can differ from the range specified for the Columns of Samples to Transfer value, but it must contain the same number of full columns. If not, and the software displays an error message, and the protocol automatically aborts. When using bare probes, use the default range of 1 to 12. The AssayMAP Bravo head moves as if it is transferring liquid from all 96 wells. The wells that contain liquid dictate which wells are transferred. Default: 1 to 12 Range: • Starting column: 1–12 • Ending column: 1–12
Initial Well Volume	The volume of liquid present in each well of the Destination Plate (deck location 7) before starting the transfer. Note: A well is defined as an individual well in a 96-well plate. But in labware where multiple probes can pull from the same continuous liquid source, a well is defined as that continuous liquid source. For example, a 12-Column Low-Profile Reservoir has 12 wells, an 8-Row Low-Profile PolyPro Reservoir has 8 wells, and a Seahorse 201254-100, PP, No Walls, Pyramid-bottom Reservoir has only 1 well. This value is used to calculate the appropriate height for the pipette tips or probes during the transfer dispense process, and to facilitate proper dynamic tip behavior. Default: 0 (µL) Range: 0–1500 (µL)
Mix Cycles after Transfer	The number of aspirate-and-dispense cycles used to mix the contents of the Destination Plate (deck location 8) after the transfer has been completed. The volume used for the mixing process is automatically calculated based on practical low-volume pipetting limits and maximum syringe or probe volume capacities. Default: 0 Range: 0–20 Mixing formula: If (x – 50 µL) > Syringe/Tip Capacity, then Mixing Volume = Syringe/Tip Capacity If 50 µL ≤ x ≤ Syringe/Tip Capacity, then Mixing Volume = 0.75x If x < 50 µL, then Mixing Volume = 0.5x where, x is the final volume in the destination wells, maximum Syringe Capacity = 250µL, maximum Tip Capacity = 140µL Note: Syringe and pipette tip capacities vary depending on the Blowout Volume setting.

d	Wash Settings

 

Property	Description
Initial Syringe/Tip Wash Cycles	The number of syringe or pipette tip wash cycles to be performed at the wash station (deck location 1) before starting the reagent transfer portions of the run. Default: 0 Range: 0–10
Final Syringe/Tip Wash Cycles	The number of syringe or pipette tip wash cycles to be performed at the wash station (deck location 1) after completing the reagent transfer portions of the run. Default: 0 Range: 0–10

e	Liquid Handling Preferences

 

Property	Description
Syringe Drying Cycles	The number of syringe purge cycles to be conducted with air before starting the run. This step occurs at different times for runs that use pipette tips instead of probes. • If using pipette tips. This step occurs before the Initial Wash cycles. • If using bare probes. This step occurs after the Initial Wash Cycles are completed. Default: 2 Range: 0–5
Pre-Wet Cycles	The number of times to wet the probes or pipette tips with the liquid from the Source Plate (deck location 7) before starting the transfer process. Note: Prewetting the pipette tips is a common pipetting technique that can greatly increase accuracy in certain situations. Default: 0 Range: 0–5
Reverse-Pipetting Overage	Specifies whether to run in reverse pipetting mode, and if so, the volume to be added to the volume that is being transferred. • To specify forward pipetting mode. Use the default setting of 0 µL. • To specify reverse pipetting mode. Use a value that is greater than 0 µL, and select the discard location for the extra liquid. This extra liquid remains in the pipette tips or probes for the entire reagent transfer process. Before conducting the mix cycles after the transfer, the excess volume is discarded to the Source Plate (deck location 7) or to Wash Station Waste (deck location 1), depending your selection. Note: Reverse pipetting is a common pipetting technique that can greatly increase accuracy in certain situations. Default: 0 (µL) Range: 0–50 (µL)
Blowout Volume	The volume of air to dispense out of the syringes or pipette tips after completing the liquid transfer steps. The blowout process is different for the reverse-pipetting and forward-pipetting modes because of the presence of the Reverse-Pipetting Overage volume. • Forward pipetting mode. The blowout occurs after the transfer has completed, and then again after mixing, if conducted. • Reverse pipetting mode. The blowout occurs only after the mixing step, if conducted, because of the presence of the Reverse-Pipetting Overage volume. Default: 0 (µL) Range: 0–50 (µL)
Liquid Class	The pipetting speed and accuracy. To ensure consistent pipetting, always select a liquid class for liquid-handling tasks. Choose from the following options: • <Automatic> (default). Automatically assigns one of the following liquid classes, based on whether pipette tips are used and the volume being transferred. These are good general-purpose liquid classes for most reagents and buffers that are used for the AssayMAP system. – Bare probes: 0–20 µL (AM_ProbesLowVol) > 20 µL (AM_ProbesHighVol) – Pipette tips: 0–20 µL (AM_250uLTipsLowVol) > 20 µL (AM_250uLTipsHighVol) • Slow Flow (5 µL/sec). A slower flow rate is better for viscous solutions. • Fast Flow (100 µL/sec). A faster flow rate is a good starting point for high organic solutions. If these flow rates do not provide the desired performance, you may enter a custom liquid class. To create a custom liquid class, you use the VWorks Liquid Library Editor. For details on how to open the VWorks Liquid Library Editor, see Creating a custom liquid class. To enter a custom liquid class for the reagent transfer: In the Liquid Class box, type the liquid class name exactly as it appears in the VWorks Liquid Library Editor.

3	In the Labware Table, select that labware you are using at deck locations 7 and 8.

 

Incorrect labware selections can cause a hardware collision, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck.

 

4	To save a new or edited method:

a	In the Save Method area, specify the new Name. If applicable, select Overwrite if Name Exists.

b	Click Save to save the settings in a method file.

 

 

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Running the Reagent Transfer utility

To run the Reagent Transfer utility:

1	If you have not done so already, select the Reagent Transfer method. For details, see Setting up the method.

2	Place the labware that you selected in your method for the Source Plate and Destination Plate at the corresponding Bravo deck locations, as shown in the Deck Layout area.

3	If you selected Use Pipette Tips, do one of the following:

•	If you selected the Transfer Pipette Tips from Deck Location 6 check box, ensure that the:

–	Pipette tip box containing the specified range of pipette tips is in position at deck location 6.

–	Empty 96AM Cartridge & Tip Seating Station is in place at deck location 2.

•	If you cleared the Transfer Pipette Tips from Deck Location 6 check box, ensure that the 96AM Cartridge & Tip Seating Station at deck location 2 contains the specified range of pipette tips.

Note: A message will appear and ask you to verify that the pipette tips are in the correct locations. Ensure that the pipette tips are arranged in the specified locations.

4	Ensure that the labware are properly seated in the platepads.

 

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.

 

5	Click Run Protocol to start the protocol run.

If the software detects a conflict in the setup, an error message or warning will appear. The following figure shows examples of Error messages that prevent the run execution.

Figure. Error message examples

 

If an Error message appears:

a	Follow the on-screen instructions to click Continue to abort the run.

b	Resolve the conflict described in the error message, for example:

–	Insufficient number of columns of pipette tips

–	Unequal number of columns selected for Source and Destination plates

–	Incompatible labware selections (labware that require pipette tips)

c	After you have resolved the conflict, click Run Protocol to start the run.

The following figure shows examples of warning messages, which allow you to continue with the run as specified, while assuming the risk posed by the warning.

Figure. Warning message examples

 

If a Warning or Verification message appears, follow the on-screen instructions verify that the settings are correct, and then do one of the following:

•	If the settings are correct, click Continue to start the run.

Note: A Pipette Tip Verification message appears whenever pipette tips are used.

•	If you need to change a setting:

–	Click Pause and Diagnose. The Scheduler Paused dialog box opens.

–	In the Scheduler Paused dialog box, click Abort process.

 

–	Change the settings to resolve the conflict.

–	Click Run Protocol to start the run.

Monitoring the run

You can monitor the progress of the run in the Status box in the upper right corner of the form.

When the run is finished, remove the labware from the deck.

About stopping or pausing the run

 

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.

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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Creating a custom liquid class

To create a custom liquid class:

1	In the Reagent Transfer v2.1 form, click Toggle Full Screen to change the display of the form so that the VWorks menubar and toolbar are visible, as the following figure shows.

2	In the VWorks window, click Tools > Liquid Library Editor. The Liquid Library Editor opens.

3	For instructions on how to use the Liquid Library Editor, see the section on specifying pipette speed and accuracy in the VWorks Automation Control Setup Guide in the VWorks Knowledge Base.

Figure. VWorks Tools menu and form displayed with full screen off

 

Figure. VWorks Liquid Library Editor window

 

 

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Automation movements during the protocol

The following table describes the basic movements of the AssayMAP Bravo Platform when you run the default Reagent Transfer utility. Different Application Settings will alter the movements.

 

Step	Action	Notes
1	The syringes are washed at the wash station (deck location 1) for the number of cycles set in Initial Syringe/Tip Wash Cycles setting.	If pipette tips are used for the transfer, this step occurs after step 3 below to facilitate pipette tip washing.
2	The Bravo 96AM Head moves above the wash station at deck location 1, and then purges the syringe contents to waste for the number of cycles set in the Syringe Drying Cycles setting.	This step is necessary because the Bravo 96AM Head is regularly used to aspirate liquids directly into the syringes, and the presence of residual liquid can cause contamination or inaccurate pipetting behavior.
3	The head picks up the pipette tips from the source tip box at deck location 6 and transfers them to the 96AM Cartridge & Tip Seating Station at deck location 2.	This step occurs only if the Use Pipette Tips check box and the Transfer Pipette Tips from Deck Location 6 check box are selected.
4	The head picks up all the pipette tips from the 96AM Cartridge & Tip Seating Station at deck location 2.	This step occurs only if the Use Pipette Tips check box is selected and the Transfer Pipette Tips from Deck Location 6 check box is cleared. A message will appear and ask you to verify that the pipette tip locations are correct.
5	The pipette tips or probes slowly aspirate and dispense the liquid in the Source Plate at deck location 7 to wet the tips or probes for the number of cycles set in the Pre-Wet Cycles setting.	This step ensures proper volume transfer for liquids with high viscosities or differences in surface tension.
6	The pipette tips or probes aspirate the volume to be transferred from the Source Plate at deck location 7.	–
7	The liquid is dispensed into the Destination Plate at deck location 8.	–
8	Steps 6 and 7 repeat until the full volume specified in the Volume to Transfer setting has been transferred to the Destination Plate.	Pipette tip maximum volume: ~140 µL can be pulled into a mounted tip before the excess fluid overflows into the syringe, because the probe protrudes into the tip volume capacity. Probe maximum volume: 250 µL can be aspirated into the syringe before the syringe is full. The probe maximum volume will be reduced automatically by an amount that is equal to the specified Blowout Volume.
9	The head performs the specified number of aspirate-and-dispense cycles to mix the new contents of the Destination Plate.	–
10	The head moves so that the pipette tips or probes are slightly above the liquid level of the Destination Plate, and then dispenses the air volume set in the Blowout Volume setting to clear any remaining liquid.	–
11	The head performs a tip touch on the left and right sides of the wells in the Destination Plate.	–
12	The pipette tips or probes are washed at the wash station (deck location 1) for the number of cycles set in the Final Syringe/Tip Wash Cycles setting.	–
13	The pipette tips are ejected to the Tip Seating Station at deck location 2.	This step occurs only if the Use Pipette Tips check box is selected.
14	The head moves above the wash station at deck location 1.	–

 

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