The serial dilution action is a stepwise dilution of a sample reagent with a buffer reagent.
For the serial dilution, it is not required that all reagents present at the start have the same concentration units.
During this action, the buffer and sample are combined and diluted by transferring a predetermined amount of sample/reagent from one container to another until the desired concentration is reached. A one-step serial dilution, can be considered as a simple dilution and you can choose the target concentration you want to obtain.
OneLab has a wizard, guiding you through several simple steps to obtain the desired dilution factor (or dilution ratio: total volume / sample volume). You only have to indicate how much volume of the buffer must be used during each dilution step and how you want to dilute the source reagent. OneLab will then automatically calculate how much sample is required to perform this action. You can also decide to choose the target concentration and volume, such that the amount of buffer reagent and amount of original sample reagent are automatically calculated. The software will also automatically tell you if it is possible to execute the desired action based on the limits of the volumes in the different labware.
The starting point depends on the application. Here, three examples will be discussed, each having its own starting layout.
There are some restrictions:
The starting point depends on the application and there are some restrictions:
- Destination labware needs to be empty
- With single sample dilutions (simple dilutions), you can add a labware with multiple locations, but are only allowed to select a single well as a source well/tube.
- When you choose to dilute a column of 8 wells with (not necessarily different) samples the labware you are allowed to use are limited to SBS format and reservoirs, where 8 channel pipettes can insert all 8 tips simultaneously and properly during aspiration and dispensing.
- You have to be sure that sufficient and appropriate labware are added to the virtual bench (see the article “Adding labware to the virtual bench”).
Example: Sample Dilution in a microplate
You need to add in your virtual workspace the labware that is involved: sources, buffer and destinations. In this example a microplate with the sample and a 50 mL conical Falcon tube of buffer 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 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:
There are two main dilution modes (indicated by “1” in the figure below). 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 have to be dragged to the top field, as indicated in the figure below (#2, 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 below).
The destinations have to be on an empty labware, in this example the microplate 96 was dragged to the bottom field on the right side (#2, bottom arrow, in the figure below).
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):
Once you have the labware dragged into the correct locations, click the green ‘Continue’ button on the top right.
During the next step you need to indicate what you exactly want to do and obtain. You can also overwrite your previous choice of “One sample” by “A column of 8 samples” or vice versa. 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):
- The dilution buffer volume (in uL)
- 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 ellipse below), including the field with the final concentration and the final volume (vertical red arrows at the bottom of the figure below):
Now you 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 to 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”.
OneLab takes into consideration the maximum allowed volume in the labware destination and warns you with a popup message if the maximum volume is reached or nearly reached.
Finally, this serial 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 blue arrow in the figure above indicates that liquid will be transferred from the labware containing the buffer reagent to the labware containing the resulting diluted sample(s). The serial dilution step (and applicable volume warnings) are indicated as a step of the protocol on the right side on the Steps tab. By double-clicking on the labware you can check the volumes and concentrations during each protocol step (see the article “Check volumes and concentrations”).
After you double click the labware, or the ‘inspect wells’ button above the microplate the wells will now have the volumes of each reagent (see image above). The well that will contain the final diluted sample has the concentration of the sample and buffer shown on the bottom of the screen, as per the Serial Dilution table from the ‘Configuration’ screen.
For more examples see the article: ‘Serial Dilution Examples’.