Setting up a Serial Dilution method

The Serial Dilution Method Setup Tool is a Microsoft Excel-based tool that uses formulas to calculate volume requirements for the samples and diluent based on your input.

The Method Setup Tool steps you through the process of defining a serial dilution method and creating a method file. The method file contains all the required information for the Serial Dilution protocol on the AssayMAP Bravo Platform. The file is a record of the intended serial dilution process.

Opening the Method Setup Tool

To open the Method Setup Tool:

1	Locate the Serial Dilution banner in the Utility Library, and then click Method Setup Tool.

 

Microsoft Excel starts and displays the Method Setup Tool.

 

 

In Microsoft Excel, ensure that you enable content.

 

The Method Setup Tool has 7 distinct steps, which are categorized as follows:

 

Step	Category	Comments
1	Setting the dilution parameters and liquid-handling options.	You define the following: • Starting concentration of the serial dilution • Optional volume to be added after running the Serial Dilution utility • Final volume of the dilution steps • Direction of the dilution series • Number of dilution step replicates • Number of mix cycles for each dilution step • Whether to use a new tip for each dilution step • Whether to prewet the tips before use • Whether to do a blowout • Which liquid class to use • Which labware to use The serial dilution plate and diluent reservoir must be an approved labware. For the labware options, see Labware.
2	Defining the dilution concentrations	You enter the final target concentration for each serial dilution step.
3	Selecting the well locations in the serial dilution plate	You select the starting well in the serial dilution plate, and the setup tool displays the plate layout based on the input provided in steps 1 and 2.
4	Calculating the sample and diluent volumes	The setup tool calculates the required volumes of sample and diluent based on the input provided in steps 1 to 3.
5	Correcting the errors.	The setup tool highlights any errors that require fixing before you can proceed to the next step.
6	Creating the method file.	The setup tool creates a comma-separated value (.csv) text file that you use to run the utility.
7	Preparing the serial dilution plates.	The setup tool provides instructions for preparing the serial dilution and diluent plates for the serial dilution utility.

 

If you change anything in step 1 or 2 after completing step 3 or 4, make sure that you click Clear Steps 3 and 4 Only and redo steps 3 and 4.

 

 

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Step 1) Set dilution parameters

Note: Only the cells highlighted in green are editable.

To set the dilution parameters:

1	To clear any data from the Serial Dilution Method Setup Tool, click Clear All (upper right corner).

2	Specify the following values in the green cells:

For additional details on these settings, see Assay development guidelines.

 

Input	Description
Final concentration of dilution 1
Concentration units	In the Input column, verify that the unit of measure (mg/mL) for the sample concentration is correct. If necessary, type the correct units. This input is used to populate other sections of the form. This value has no impact on the subsequent data or volume calculations.
Concentration value	In the Value column, type the final concentration of the most concentrated level in the serial dilution series (e.g., Dilution 1 in Step 2).
Optional volume (µL) to be added	The volume to be added manually or using the Reformatting utility after the Serial Dilution run, for example, an internal standard. The set up tool uses this value to reduce the volume of the serial dilution to the final volume minus the optional volume to be added. The set up tool also uses this value to increase the concentration of each serial dilution step (see Post Run cells in Step 2) so that when the optional volume is added, the final serial dilution volumes and concentrations match the desired final serial dilution series volumes and concentrations. IMPORTANT The value you enter here must be less than the Final volume. The recommended minimum volume is 5 µL. For example, if the Final volume = 50 µL and Optional volume to add later = 10 µL, the Serial Dilution steps will have a volume of 40 µL after the run, at which point the optional 10 µL will be added for a total volume of 50 µL. Default: 0 (µL) Range: 0–50 (µL)
Final volume (µL) of dilution series	The final volume of each step in the dilution series. This includes the Optional volume (µL) to be added. Note: The practical range is approximately 20–500 µL, depending on the number and concentration of the dilution steps. The high end of the range is limited by well capacities and the high volume transfers required during the early steps of the dilution series compared to the final volume of the dilution steps. The low end of the range is limited by the requirement that the transfers be 5 µL or greater to ensure high precision and accuracy. Large-volume dilution series require more mix cycles (up to 100 for the largest volume serial dilutions) because of the limited volume that can be used for each mix cycle. The results for large volume dilution series may not be as accurate as smaller volume dilution series. Default: 0 (µL) Range: 5–500 (µL)
Direction of dilution	The direction in which the dilution series should progress. The dilution series follows a serpentine pattern. See the example in Step 3) Select the starting well of the dilution series. Default: top to bottom Options: top to bottom, bottom to top, left to right, right to left
Number of replicates	The number of replicates of each dilution step in the dilution series. Note: This cell turns yellow if you select 5 replicates. The yellow cautions you that the serial dilution may not fit on the serial dilution plate, depending on the number of steps in the dilution series. The maximum number of dilution steps with 5 replicates is as follows: • 16 for top-to-bottom or bottom-to-top dilution series • 12 for left-to-right or right-to-left dilution series Default: 1 Range: 1–5
Mix cycles after each dilution	The number of aspirate-and-dispense cycles used to mix the contents of the wells in the serial dilution plate. Default: 10 Range: 0–100
Change pipette tips after each dilution	The option to change pipette tips before transferring the sample for each dilution step. Default: Yes Options: Yes, No
Pre-wet pipette tips before transfer	The option to wet the pipette tips with the sample before aspirating the sample. Note: Prewetting the pipette tips is a common pipetting technique that can increase accuracy in certain situations. Default: Yes Options: Yes, No
Blowout volume (µL)	The volume of air to be drawn into the pipette tips before aspirating the sample. After dispensing the transferred sample, the volume of air remaining in the tips is dispensed (blown out) while the tips are still in the wells. The blowout is followed by a tip touch on the east and west sides of the well. If the Blowout volume is set to 0, no blowout or tip touches will occur. Default: 5 (µL) Range: 0–50 (µL)
Liquid class	The pipetting parameters (for example, aspiration and dispense speed). The liquid class selection strongly influences the pipetting precision and accuracy. This choice only controls the aspiration and dispense of the samples and diluent. The mixing speed of the dilutions is fixed at 300 µL/sec. Options: • <Automatic> (default). Automatically assigns one of the following liquid classes, based on the volume being transferred. 0–20 µL (AM_250uLTipsLowVol) > 20 µL (AM_250uLTipsHighVol) These are good general-purpose liquid classes for most reagents that are used with the AssayMAP system. • Slow Flow (5 µL/sec). A slower flow rate is better for viscous solutions. • Fast Flow (100 µL/sec). A faster flow rate may improve performance 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 To create a custom liquid class:. To enter a custom liquid class: In the Liquid Class box, type the liquid class name exactly as it appears in the VWorks Liquid Library Editor. Note: The estimated run time (Status box area) may not be accurate for methods that use a custom liquid class.
Serial dilution plate	The labware that you are placing on the plate riser at deck location 6. For options, see Labware. For the capacity limits and the volume overage recommended for each labware, see the Labware Reference Guide in the Literature Library page of the workbench. Note: The intermediate volumes required to generate the serial dilution will be larger than the final volume of the serial dilution. If you have chosen a plate type whose capacity is lower than required, you will be alerted in steps 4 and 5.
Diluent reservoir	The labware that you are placing on the plate riser at deck location 2. For options, see Labware. For the capacity limits and the volume overage recommended for each labware, see the Labware Reference Guide in the Literature Library page of the workbench. Be sure to choose a diluent reservoir that can hold the volume required for the serial dilution. The volume of diluent and the location where it should be placed will given in step 7 of the Method Setup Tool. The plate type chosen must have enough well capacity to hold the dilution indicated in step 7 plus the required well overage. For volume guidelines, see Preparing the diluent and samples. Note: If the selected labware cannot hold the volume of diluent required for the serial dilution, an error message will appear in steps 4 and 5.

Pipetting accuracy and liquid classes

 

Agilent Technologies does not guarantee specific accuracy and precision results for the Serial Dilution v2.0 utility because accuracy and precision are too dependent on the composition and liquid properties of the solutions used.

 

Pipetting accuracy and precision can be greatly affected by liquid properties, such as viscosity and surface tension. If unacceptable accuracy and precision results are observed, you may modify these liquid classes to achieve acceptable results. Use the following procedure to modify or create a liquid class.

 

To create a custom liquid class:

1	In the Serial Dilution 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.

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

 

Figure. VWorks Liquid Library Editor window

 

 

Step 2) Define dilution concentrations

To define the concentration of the serial dilution steps:

In the green boxes in the Final column, enter the desired concentration of each serial dilution step.

The method setup tool automatically enters the dilution concentration for dilution 1 when you enter the Final concentration of dilution 1 (Step 1) Set dilution parameters). You enter all the other concentrations in the dilution series directly in each cell or by entering a formula in the cell. You can use the Excel fill series function with formulas to quickly generate a serial dilution.

You can generate serial dilutions with 2 to 24 steps. You can also generate blanks by putting zero as a dilution concentration.

Note: If you enter zero as a concentration, an error message appears and says Negative or zero values cannot be plotted correctly on log charts. Only positive values can be interpreted on a logarithmic scale. To continue and use the zero, click OK. The graph will not include the zero volume but the method will work.

The Post Run column contains non-editable cells that show the concentration of the serial dilution series after the run is complete.

•

If step 1 Optional volume to be added is >0, the Post Run column will show the concentration of the serial dilution steps after the serial dilution utility is run but before the optional volume is added. The concentrations entered in the Final column represent the concentration of the serial dilution after adding the optional volume.

•	If step 1 Optional volume to be added is 0, the concentrations entered in the Final column will be the same as the those in the Post Run column.

As you define the concentrations, the graph displays the corresponding log-log plot of the dilution series concentrations. You can use the graph to verify that your dilution formula will yield the expected results. The graph can help you visualize the distribution curve and identify gaps between dilution steps.

 

 

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Step 3) Select the starting well of the dilution series

 

If you change anything in step 1 or 2 after starting step 3 or 4, make sure that you click Clear Steps 3 and 4 Only and redo steps 3 and 4.

 

To define the layout of the serial dilution plate:

1	Click Select Starting Well (figure, item 1) and then click the corresponding cell in the Serial Dilution Plate map where the replicate 1 of dilution 1 will be located (figure, item 2).

 

2	When the Choose Starting Location dialog box opens, displaying a formula, click OK (figure, item 3).

The layout for the dilution series appears in the Serial Dilution Plate map, displaying the selected number of replicates in the specified layout (row or column).

Figure. Example of a left-to-right layout by row for a dilution series with 4 replicates

 

Figure. Example of a top-to-bottom layout by column for a dilution series with 4 replicates

 

 

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Step 4) Calculate the sample and diluent volumes

To calculate the sample and diluent volumes:

Click Calculate Volumes. The tool uses the information provided in the previous steps to calculate the sample and diluent volumes that will be transferred into each well to achieve the specified serial dilution series.

The calculated volumes (µL) appear in the following areas:

•	Sample Volume plate map

•	Diluent Volume plate map

 

 

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Step 5) Correct these errors

Although you may enter values that are not possible or practical given the selected labware or specified concentrations and volumes, the setup tool will flag such values as errors. You must resolve any errors before you can proceed to the next step in the setup tool.

 

If you change anything in step 1 or 2 at this point, click Clear Steps 3 and 4 Only and redo steps 3 and 4.

 

 

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Step 6) Create the method file

To create the method file for serial dilution:

1	Click Create Serial Dilution Method.

2	In the Save As dialog box, specify the file name and the storage location, and then click Save.

The setup tool saves the file as a comma-separated value (.csv) text file.

The default method file storage location is in the following folder:

… /VWorks Workspace/Methods/AM Serial Dilution Utility v2.0

 

 

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Step 7) Prepare serial dilution plates

To prepare the serial dilution plates:

1	Follow the instructions that appear under step 7 to prepare the serial dilution plates.

2	To close the Method Setup Tool, click File > Close or exit the Microsoft Excel software.

A Microsoft Excel dialog box opens and asks if you want to save the changes. You may click Don’t Save.

 

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