Reagent Aliquot v1.1

The Reagent Aliquot utility aliquots a reagent from a single column or reservoir of a Bulk Reagent storage plate into 1 to 12 columns of a 96-well microplate (Reagent Aliquot plate). The utility uses a single column of eight Agilent 250-µL pipette tips to prepare the aliquots.

This utility is a useful starting point for most applications and workflows, because it simplifies the process of preparing reagent plates for an automation run.

Before you start

•	Ensure that you have properly planned the automation run. Make sure to use the application-specific Reagent Volume Calculator to:

–	Determine the amount of reagent or sample required in each well.

–	Make appropriate labware selections.

–	Calculate bulk reagent volumes required, including required overage volumes.

•	Prepare appropriate volumes of bulk reagents, and manually prepare a Bulk Reagent Plate.

•	Prepare the Bulk Reagent Plate with the intended reagents. Pipette an excess volume in the plate of at least:

–	PCR plates: 15–20 µL per well

–	U-bottom, V-bottom, and flat-bottom microplates: 25–30 µL per well

–	Deep-well plates: 35–45 µL per well

Note: These overage volumes are higher than in other applications because this utility uses reverse pipetting.

 

A small reagent volume excess is required in all labware types to ensure proper volume transfer. Look up the recommended values for each labware type in the Labware Reference Guide.

 

Note: You can find the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Accessing the Reagent Aliquot utility

To access the Reagent Aliquot utility:

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

 

The following figure shows the Reagent Aliquot utility interface.

 

 

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Running the Reagent Aliquot utility

To run the utility:

1	In the Application Settings area of the Reagent Aliquot utility, specify values for the following settings:

 

Setting	Description
Columns of Pipette Tips in the Pipette Tip Box	Specifies the range of pipette tips present in the Pipette Tip Box before the run starts. • In the left box, specify the column number of the first column of pipette tips present in the tip box. • In the right box, specify the column number of the last column of pipette tips present in the tip box. One full column of pipette tips is used for every run of this utility. The remaining pipette tips are automatically returned to the Pipette Tip Box for use in future runs. IMPORTANT The Pipette Tip Box must contain contiguous columns of 8 pipette tips each, within the range of columns specified. CAUTION A collision can occur if any pipette tips are present in the columns outside the specified range.
Columns To Receive Aliquots in the Reagent Aliquot Plate	Specifies the range of wells that you want to fill in the Reagent Aliquot Plate. • In the left box, specify the column number of the first column in the Reagent Aliquot Plate that will be filled with aliquots. • In the right box, specify the column number of the last column in the Reagent Aliquot Plate that will be filled with aliquots.
Aliquot Reagent Source Column from the Bulk Reagent Plate	Defines the column of the Bulk Reagent Plate that contains the bulk reagent to be aliquoted into the Reagent Aliquot Plate.
Aliquot Volume (µL)	Defines the volume of bulk reagent to be aliquoted to each of the selected wells in the Reagent Aliquot Plate.

2	In the Labware Table, select the labware that you are using for the Reagent Aliquot Plate (deck location 4) and the Bulk Reagent Plate (deck location 7).

 

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.

 

3	Place the reagent plates, tip box, and 96AM Cartridge & Tip Seating Station on the AssayMAP Bravo deck. Confirm that the physical layout matches the Deck Layout and Labware Table sections of the interface.

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.

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

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

 

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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Automation movements during the protocol

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

 

Step	Action	Notes
1	The Bravo 96AM Head moves above the wash station at deck location 1, and purges the syringe contents to waste.	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.
2	The head picks up all the pipette tips from the Pipette Tip Box at deck location 6.	Although all the pipette tips are picked up from the tip box at this step, only 1 column of pipette tips is used for this utility.
3	The head ejects the pipette tips into the 96AM Cartridge & Tip Seating Station at deck location 2.	The Bravo 96AM Head is centered over the Tip Seating Station when ejecting the tips.
4	If applicable, the head picks up any columns of excess tips from the Tip Seating Station at deck location 2.	If this step is required, the Bravo 96AM Head will offset to the right side of the Tip Seating Station when picking up columns of excess pipette tips.
5	If applicable, the excess tips are ejected back into the Pipette Tip Box at deck location 6.	A single column of pipette tips is used for this utility. During this step, any excess tips are returned to the Pipette Tip Box for use in future processes. Excess tips are always returned to the left side of the Pipette Tip Box, starting at column 1.
6	The head picks up a single column of tips from the Tip Seating Station at deck location 2.	–
7	The bulk reagent is aspirated into the tips from the Bulk Reagent Plate at deck location 7.	This step uses reverse pipetting, which involves aspirating a small excess of liquid into the tips to ensure accurate pipetting for the final aliquot of a set.
8	The bulk reagent is dispensed into the Reagent Aliquot Plate at deck location 4.	The utility uses either of the following two dispense modes: • Multiple low-volume aliquots are dispensed from a single aspirate step. • A single high-volume aliquot requires multiple aspirate steps. In both cases, a small excess volume of the bulk reagent remains in the tips because of the reverse pipetting technique used.
9	Step 7 and Step 8 are repeated until all aliquots have been prepared in the Reagent Aliquot Plate.	–
10	Excess reagent within the tips is dispensed back into the Bulk Reagent Plate.	–
11	The single column of used tips is ejected to the Tip Seating Station at deck location 2.	The used tips are always ejected to column 1 of the Tip Seating Station.
12	The head moves above the wash station at deck location 1.	–

 

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