Here, three examples of Serial Dilutions will be discussed, each making use of different labware. Please reference the article called ‘Serial Dilutions’ for and explanation and the example of diluting a well in a microplate. For these examples, your reagents need to be present at the start of the protocol (see the article “Fill a labware with a reagent” to add reagent to the labware on the virtual bench).

Example 1: A simple dilution -> the sample at the start will be diluted with buffer reagent in a new labware location to the exact desired concentration.

Example 2: A single channel serial dilution -> the sample at the start, present in a single labware location, will be diluted with buffer reagent in several new labware locations, creating a series of diluted samples with different concentrations.

Example 3: An 8 channel serial dilution -> the samples at the start are present in a column of 8 wells of a 96-well microplate. They will be diluted with buffer reagent in several new labware locations, creating a series of diluted samples with different concentrations for each of the starting samples.

 

Example 1: A simple dilution.

You need to select the labware that is involved: sources, buffer and destinations. In this example three identical 50 mL conical Falcon tubes are used. The names of each labware are updated to be convenient, see the article “The labware contextual menu” for more information. The starting conditions for this first example are as follows:

After that, you can add the serial dilution step to the list of steps that need to be executed during the protocol. Click on the “Actions” menu button in the Protocol Menu Bar on the left of your screen. The “Actions” menu will pop-up. Then, click on the “Serial dilution” option:

The “Serial dilution” menu will appear:

There are two main dilution modes (indicated by “1” in the figure). The first one makes use of a single sample to start with. The second one uses a column of 8 samples that will be diluted.

The labware locations that act as source need to be dragged to the top field, as indicated in the figure above (#2 in the figure, top arrow). If the first mode is selected, where only one sample will be diluted, you can only drag a single location labware to this field. If you try to add a second labware, you will receive a warning in a popup stating: “Only one labware is required.” Please remove the current labware before selecting a new one”. The original sample can have any concentration units.

The reagent that acts as the dilution buffer needs to be present in a single location. The reagent in this labware can be of arbitrary choice: the name and concentration are not important, but the volume needs to be sufficient to be able to execute the entire action. You need to drag this labware to the field in the middle (#2, center arrow, in the figure above). 

The destinations have to be on an empty labware, in this example an empty 50mL conical Falcon tube was dragged to the bottom field on the right side (#2, bottom arrow, in the figure above).

In case you made a mistake, you can remove the undesired labware and drag the desired labware to the field you want during a retry (red circles in following figure):

During the next step you need to indicate what exactly you want to do and obtain. You can also overwrite your previous choice of “One sample” by “A column of 8 samples” or vice versa, indicated by the red arrows in the following figure. On the left side, you can still change the labware used as source, buffer and destination by clicking the ‘Edit Selection’ button.

The steps you have to fill out in this menu are similar to the “Single pipetting action”. Including several sections: “Configuration”, “Advanced”, “Automation” and “Guidelines” (see also the “Pipetting menu” article). Changing tips between pipetting steps and the position of the end of the tip are important parameters, since concentrations are involved, contamination needs to be considered. There are dedicated articles by those names explaining what each setting means.

In this first example, we are aiming for a “Desired final concentration” in the destination location. However, this field is not displayed at this moment: First you have to indicate two important parameters (horizontal red arrows in the following figure):

  1. The dilution buffer volume (in uL)
  2. Dilution factor (with a minimum of 1.1) There is a special checkbox to be able to set the value directly to 3.16 (half-log).

After you set your desired volume and dilution factor, the result of the simple dilution is displayed (in the red circle below), including the field with the final concentration and the final volume (vertical red arrows):

Now that the displayed menu updated, you actually have more settings you can change to obtain the diluted reagent that you desire. The sample concentration at the start is fixed and can only be changed by replacing the “reagent at start”, the sample volume that is required will vary according the settings of the following four fields:

  • “Dilution buffer volume”
  • “Dilution factor”
  • “Final volume”
  • “Final concentration”

The relations between these variables are as follows:

  • “sample volume” = “dilution buffer volume” / (“dilution factor” – 1)
  • “final volume” = “sample volume” + “dilution buffer volume”
  • “final concentration” = “sample concentration” / “dilution factor”

Therefore, once you filled out a value for the “dilution buffer volume”, all other variables are updated directly, taking into consideration the default value of 2 for the “dilution factor”.

Now, you can change the values of each of the fields according to your wishes. Changing them has the following impact:

  • Changing the “dilution buffer volume” will recalculate the “sample volume” and update the “final volume” accordingly
  • Changing the “dilution factor” will recalculate the “sample volume” and update the “final volume” and the “final concentration” accordingly.

Because of the relation between the several variables, changing the values in the two last fields will logically have a similar (but reversed) impact.

  • Changing the “final volume” will recalculate the “sample volume” and update the “dilution buffer volume” accordingly.
  • Changing the “final concentration” will recalculate the “sample volume” and update the “final volume” and the “dilution factor” accordingly.

OneLab takes into consideration the maximum allowed volume in the labware destination and warns you with a popup message if the maximum volume is (b)reached.

To keep the numbers simple and straightforward for this example, the fields are updated such that a “dilution factor” of 4 is present. The original “sample concentration” is 512 mM, so the “final concentration” will be 128 mM. And the “dilution buffer volume” is chosen to be 30000 uL (30 mL), such that the “sample volume” is simply calculated to be 10 mL and the “final volume” 40000 uL (40 mL). Now you can set all the specific pipetting parameters by scrolling down and choosing the options you like. Finally, this simple dilution step is implemented when you click the green “Save step” button at the top right.

The resulting protocol step is then automatically generated by Onelab:

The arrows indicate that reagent is transferred from the labware containing the original sample and the one containing the buffer reagent to the labware containing the resulting diluted sample. The serial dilution step is indicated as a step of the protocol on the right side on the Steps tab. By clicking on the labware you can check the volumes and concentrations during each protocol step (see the article “Check volumes and concentrations”). Here the labware containing the original sample will indicate that the reagent has a concentration of 512 mM. The volume is set to “AUTO”.

In the labware that will contain the final diluted sample, the concentration of the sample is reduced and is now 128 mM. The concentration of the buffer reagent is reduced to 0.75 a.u. (arbitrary units) as it started as 1 a.u.

To conclude: by selecting a single location as destination, the serial dilution is reduced to a simple dilution where you have the option to set the final volume and final concentration (with the restriction that you know the sample concentration at the start).

Example 2: A single channel serial dilution

The starting conditions for this second example are different from the first example. Here, four 15mL tubes are available destinations for the serial dilution. The start is as follows:

Again, you can click on the “Actions” menu button in the Protocol Menu Bar on the left of your screen, then click on the “Serial dilution” option. The “Serial dilution” menu will appear again and you can drag and drop the labware with the original sample to the “sample to dilute” field, the labware with the buffer reagent to the “dilution buffer” field and the other four empty tubes to the “destinations” field. You have to click and drag these one by one. The order in which the labware is present in the destinations window influences the order in which the serial dilution is performed.

You have the same options here, as described in Example 1. You can change the labware you want to use and the mode and you can set the:

  • “dilution buffer volume”
  • “dilution factor”
  • “final volume”
  • “final concentration”

with the same behaviour as described in the first example. However, there is now a list/table available for each of the dilution steps. Changing the “final concentration” in any of the locations of the destination labware, will update the other values accordingly. For the “final volume” only the volume of the last location in the list can be set. The other volumes and the dilution factor are updated automatically and according to the value you put in this last field:

Clicking on the green “Save step” button will result in the following situation:

The arrows indicate that reagent is transferred from the labware containing the original sample and the one containing the buffer reagent, to the labware finally containing the diluted sample. The serial dilution step is indicated as a step of the protocol on the right side on the steps tab. By clicking on each of the individual labware you can check the volumes and concentrations during each protocol step (see the article “Check volumes and concentrations”). Note here that the protocol first allows you to dispense buffer reagent in each of the destination locations. After that, the correct volume of the original sample is taken from its labware and transferred to the first destination location. There the already present buffer reagent and the sample are mixed, according to your personalized settings. After that, the correct mixed volume is transferred to the second destination location. There the process repeats. The last step is a transfer to the last labware, notice here, that the arrows indicate from which labware to which labware each transfer takes place.

Example 3: An 8 channel serial dilution

By using an 8 channel pipette, the labware containing the buffer reagent needs to be of the “reservoir” or 96-well microplate type, such that all 8 tips can enter and aspirate the reagent properly. Therefore, in this third example, the samples at the start are present in the first column of 8 wells of a 96-well microplate. They will be diluted with buffer reagent from a reservoir, in several new labware locations (another column) on the same microplate, thus creating a series of diluted samples with different concentrations for each of the starting samples. Here you have the choice to make a one-step serial dilution (a simple dilution as explained in Example 1), or more steps if you like (by selecting multiple destinations), creating a serial dilution for each of the samples, with the same dilution factor. Not only the concentrations of the samples might vary, you have the freedom to use different units of concentration for each sample as well. In this example, the first column contains the samples, the starting situation is as follows:

Again a serial dilution action needs to be implemented as in the other examples. Click on the “Actions” menu button in the Protocol Menu Bar on the left of your screen. The “Actions” menu will pop-up. Then, click on the “Serial dilution” option. The “Serial dilution” menu will appear again. This time, drag and drop the labware with the original sample to the “sample to dilute” field, in this case, the 96-well microplate. You will be directed to the “Select samples to dilute” menu, where you need to select a column (by clicking on one of the arrows above each column). In this example, the first column contains the samples:

By clicking on the green “Save selection” button, you are brought back to the “Serial dilution” menu, where you can now drag and drop the labware containing the dilution buffer (the reservoir) and the labware with the destinations. By dragging the same 96-well microplate to the “Destinations” field (or a new labware according to your preference), a new menu appears “Select destinations”. Here you can either select a single column, to perform a simple dilution with a fixed “dilution factor” for all samples, or you select several columns, to perform a serial dilution per sample, but simultaneously when using a multichannel pipette. In this specific example, we chose for the serial dilution and select the last four columns of the microplate. Note that on the right side, the selection is indicated and the information that “Each column will be a dilution step” displayed. Additionally, the number of dilution steps are indicated (in the red circle in the figure below):

You have the same options here, as described in Example 1 and 2. You can replace the labware you want to use, you can change the mode and you can set the:

  • “dilution buffer volume”
  • “dilution factor”
  • “final volume”
  • “final concentration”

There is now a list/table available for each of the dilution steps AND you can choose your sample to dilute. This means that you can choose any of the wells of the first column, A1-H1 and see what happens during the serial dilution of each of the samples. Labware locations, concentrations, volumes and concentration units are updated accordingly. Note here, that changing the “final concentration” in any of the locations of the destination labware, will update the other values as well, since the “dilution factor” will be updated accordingly and is valid for ALL samples simultaneously.

In the following figures, different samples are selected (indicated by the red circle): the details of the serial dilution in the table are updated accordingly, i.e. the locations (wells, indicated by the red arrows), the concentrations, volumes, and concentration units (indicated by the red rectangle).

Clicking on the green “Save step” button will result in the following final situation:

Here, the arrow indicates that buffer reagent is transferred from the reservoir to the microplate. The serial dilution step is again indicated as a step of the protocol on the right side on the steps tab. By clicking on each of the individual locations of the microplate you can check the volumes and concentrations during each protocol step (see the article “Check volumes and concentrations”). OneLab automatically sets the destination order for the dilution steps in the ascending order of the columns of the microplate. In this example columns 9 through 12 were selected, so the first dilution step happens in column 9, the second in 10, the third in 11 and the fourth (and last) in column 12 will have your final concentrations.

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