The normalization planning tool helps users determine how much diluent (usually ddH2O) to add to a set of aliquots (solid or liquid) to bring them all to the same target concentration.
You can find more detailed definitions of the terms mentioned in this article in the glossary.
Input: Plates or Racks
Output: Normalization Worklists
Prerequisites:
(required) The plate(s) or rack(s) must already exist in inventory (Inventory > Plates)
If the plate does not exist yet, use the header to go to Inventory > Plates > ‘New Plate’ or ‘Upload’ to create a new plate.
(required) The materials in the plates must have either concentration or mass for the normalization calculations to be performed
The Normalization Planning tool is under Tools > Tool Library. Click ‘Launch Tool’ to start using it.
The tool will walk you through several steps to generate a worklist for normalization planning. You need to complete each step before proceeding to the next. After completing a step, the completed step mark (circle) will turn green with a checkmark. You can always use the ‘Previous’ button to go back to any previous step].
1. Select Inputs: To select plates from the plate inventory, click on ‘Select Plates’ and check the boxes next to the plates of materials that you want to normalize. Then click ‘Select Plates’ from the pop-up window to confirm your selection.
You can view the selected plate(s) in a table format. To view the content of a plate, click on the eye icon to the left of the plate’s row. To remove a single plate, click the red trash icon to the right of the row. To remove all selected plates, click the red trash icon next to the ‘Change Plates’ button. When satisfied with the selection, click ‘Normalize <plate’s name>,’ where ‘plate’s name’ is the name of the selected plate. Then, you'll see a visualization of the plate's sample with their volume and concentration.
2. Normalization Parameters: This step requires you to enter a target concentration value and specify the concentration unit for the aliquots of the selected plate. You can directly normalize on the same plate if your samples are dry. If the chosen plate contains liquid materials, there is an option to transfer some of the materials to a new plate before normalization by checking the ‘Transfer Before Normalization’ checkbox. In this case, since the volume in the wells exceeded the maximum if we normalized to 5 ng/uL, we have decided to transfer 10 uL to another plate.
You will need to specify the volume of the materials to be transferred from the selected plate to a new plate. You can give the new plate a name and generate a barcode for that new plate. However, if the materials in the selected plates are not liquid materials (samples that do not have a volume), an error will be displayed.
3. Diluent Container: This step allows you to select the type of container that stores the diluent (usually ddH2O) used to normalize the materials in the selected plate. The default diluent container is a reservoir holding up to 1L of diluent material. You can give the diluent container a name, specify its dead volume, and specify the number of diluent containers needed for normalization.
The dead volume of the diluent container is the residual volume of the diluent in the container following the transfer of liquid. It is usually a very small amount of liquid (diluent) that cannot be taken up by a liquid handler. The default dead volume is 0 mL. The number of diluent containers (default is 1) is calculated based on the type of selected reservoir and the calculated total volume of diluent required to reach the specified target concentration. These parameters help you prepare labware and set up the normalization. In most cases, you can accept the default parameters. When you are done setting up the diluent container, click ‘Next’ to review the normalization worklist.
4. Review Worklist: This step allows you to review the normalization worklist. If everything looks good, you can give the worklist a descriptive name and click ‘Save Worklist’. Otherwise, use the ‘Previous’ button to return to previous steps and make changes.
For this case, we have two worklists; one is the pre-normalization transfer worklist that will take 10 uL of samples from the source plate to the "Normalized plate primers" plate, where normalization will occur.
The second one is the normalization worklist, in which volume from the reservoir will be added to the "Normalization plate primers" plate.
You will then be notified that the normalization planning is completed and that there is a link to the normalization worklist. You can view these worklists by clicking "Normalization Worklist(s) Created: 2". Alternatively, you can access this worklist from the worklists table by going to Data>Worklists.
An example of the normalization worklist is shown below. You can export this worklist in the preferred format of different liquid handlers by clicking on ‘Export Worklist’.
After executing the worklist in the lab, use the Execute Worklist tool under Tools > Tool Library to update the plate in the inventory.
See also:
Add Reagent Tool