Preparing the reagents

The In-Solution Digestion: Single Plate application accommodates a wide range of digestion chemistries so that previously optimized conditions can be easily transferred to this automated platform. For this reason, it is not practical to describe specific reagent chemistries. Agilent Technologies recommends that you attempt to transfer your manual in-solution digestion reagent chemistries directly onto the automated platform.

When preparing bulk reagents for use in this App, the total volume required for a reagent will depend on the type of labware that is selected. Different types of labware have different well geometries, which results in variability in the amount of excess reagent volume required to ensure reliable pipetting behavior. Use the Reagent Volume Calculator for In-Solution Digestion: Single Plate to help determine the appropriate bulk reagent volume to prepare for a given experimental procedure.

 

A small reagent volume excess is required in all labware types to ensure proper volume transfer. Use the Reagent Volume Calculator to automatically include excess volume, or 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.

About the Reagent Volume Calculator for In-Solution Digestion: Single Plate

The Reagent Volume Calculator is a Microsoft Excel worksheet that automatically calculates bulk reagent preparation volumes based on the application settings.

Based on the number of samples to process, the calculator manages the dead volume, pipetting overage, and evaporation concerns for your experiment. For volume overage by labware type, see Labware.

The following figure shows the worksheet of the Reagent Volume Calculator for the In-Solution Digestion: Single Plate v1.1 application.

Figure. Reagent Volume Calculator worksheet

 

 

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Using the Reagent Volume Calculator for In-Solution Digestion, Single Plate

The color of the fields in the Calculator provide cues to identify how the data meets with expectations:

•	Green indicates a field where you enter a value. If the box stays green after you enter the value, the value meets all the requirements.

•	Yellow indicates a caution that the value meets only some of the requirements. Use caution in proceeding.

•	Red indicates a warning that the value does not meet the requirements.

If a box turns yellow (caution) or red (warning), flagging a potential issue, see the relevant Field description and value range in the following table or see the corresponding tooltip for the box.

To use the Reagent Volume Calculator:

1	Open the App Library. See Accessing an application.

2	Locate In-Solution Digestion, Single Plate, and then click Calculator. Microsoft Excel starts and displays the calculator.

3	Ensure that you enable content in Microsoft Excel.

4	In the calculator, enter the values in the green boxes (data entry fields):

a	Specify the Sample Plate Settings:

 

Setting	Description
Number of Full Columns of Samples	The number of full columns of samples that will be processed in the run and the corresponding number of columns of pipette tips present in the Cartridge & Tip Seating Station. IMPORTANT You must specify a contiguous array of full columns. The pipette tips must be in the Cartridge & Tip Seating Station in the same arrangement as the samples in the Sample plate. Note: One full column consists of 8 pipette tips. Value: • Default: 1 • Range: 1–12
Starting Sample Volume	The volume (µL) of sample present in the Sample plate at the start of the protocol. Value: • Default: 20 • Range: 0–1000

b	Specify the Addition Step Settings: Type the volume to use for each Reagent addition.

 

Setting	Description
Volume, Addition 1	The volume (µL) of reagent to be transferred from deck location 5 to the Sample plate at deck location 4. • Default: 10 • Range: 0–1000
Volume, Addition 2	The volume (µL) of reagent to be transferred from deck location 6 to the Sample plate at deck location 4. • Default: 10 • Range: 0–1000
Volume, Addition 3	The volume (µL) of reagent to be transferred from deck location 8 to the Sample plate at deck location 4. • Default: 10 • Range: 0–1000
Volume, Addition 4	The volume (µL) of reagent to be transferred from deck location 9 to the Sample plate at deck location 4. • Default: 10 • Range: 0–1000
Cumulative Sample Plate Volume	The calculated total volume in the Sample plate after adding the reagents.

c	Specify the Labware Options: The calculator will estimate the volumes required per well, including the volume of bulk solution required to fill the labware.

 

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

 

For a list of labware and the excess volume recommended per well or channel, see Labware.

 

Setting	Description
Deck Location	Displays the number of the corresponding location on the AssayMAP Bravo deck.
Reagents	Displays the type of solution at the corresponding deck location.
Labware	Specifies the labware type for the corresponding deck location. Click the green box, and then select the labware type from the list.
Excess required per well (µL)	Specifies the recommended excess volume (µL) per well or channel. By default, the Calculator uses the minimum recommended excess volume per well or channel. You may overwrite these values to adjust the volume based on the duration of the run and the reagent volatility. For the reagent plates, use the maximum well volume, if possible, to minimize any potential effect due to evaporation.
Max well volume (µL)	Displays the volume (µL) capacity per well or channel for the selected labware. This volume takes into account the displacement caused by the pipette tips.
Volume per well required (µL)	Displays the calculated volume (µL) per well or channel that must be in the labware at the beginning of the run.
Bulk volume required per well (mL)	Displays the bulk volume for the total number of wells or channels used for the run, including a 10% excess.

 

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Creating reagent plates

 

To prevent evaporation, dispense the reagents into the appropriate labware immediately before running the protocol.

 

When preparing to do a digestion run, it will often be necessary to aliquot bulk reagents into their individual reagent plates. You can aliquot the reagents using manual pipettes, or you can run the Reagent Aliquot utility to transfer the reagents automatically. For instructions, see Reagent Aliquot v1.1.

 

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Preparing the 96AM Cartridge & Tip Seating Station

If the protocol requires pipette tips, you must fill the 96AM Cartridge & Tip Seating Station with the appropriate number and configuration of pipette tips to match the samples to be processed. To prepare the pipette tips, run the Pipette Tip Transfer utility. For details, see Pipette Tip Transfer v1.1.

 

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