How do I normalize samples?

This article provides a step-by-step walkthrough of how to create a Normalization step on the Microlab Prep. Keep in mind that your Prep must have 2 Independent Channels to perform Normalization.

Normalization is available in software version 2.1 or later. Download the latest software for access to all the latest features on the Prep.

Requirements

• USB drive with worklist
• Tips (optional for deck scan)
• Source, diluent, and target labware (optional for deck scan)

Procedure

1. Tap New Protocol to get started. If you are adding a Normalization step to an existing protocol, skip to step 4.
2. If your labware is available, place it on the deck and tap Detect. If the camera is not being used to detect labware, tap the Add button to manually select each piece of labware for the deck.
3. Confirm the deck layout after making any edits needed. If your labware is not available, learn about requesting new labware.
4. When the protocol overview appears, tap New Step, Advanced, then select Normalization.
5. Select whether the step will always use the same file or prompt the user for the file at the beginning of the protocol. If the same file will be used every run, tap Browse to select the file. Make sure you have a USB drive ready with the file.
6. Check what is required for the worklist. Tap “Review file requirements” to see an example worklist. The following columns are required in every Normalization worklist:
   • SourceSite and SourceWell: The source site is the number of the deck site where the source labware containing the samples will be located (1, 2, … 8). The source well is the label of the well or tube (A1, B1, …) on the source labware to aspirate from.
     
     An optional “SourceId” column containing barcodes or IDs for each sample can also be included for traceability.
   • Concentration: The initial concentration of each sample. These values are used with the final concentration and volume (specified in steps 9 and 10) to calculate the volume of sample and diluent to pipette for each sample. The value can have any units, as long as they are all the same and the final concentration uses the same units.
   • TargetSite and TargetWell: The target site is the number of the deck site where the target labware will be located (1, 2, … 8). The target well is the label of the well or tube (A1, B1, …) on the target labware to dispense to.
7. If you have already calculated the sample and diluent volumes and included them in the worklist, enable “Samples and diluent volumes”. They must be added in columns with the headers “SampleVolume” and “DiluentVolume”, respectively. Make sure all values are in µL.
   
   When the sample and diluent volumes are included in the worklist, the concentrations are still required, but they are only used for error handling and traceability.
8. Tap Next after setting up the worklist file.
9. If your worklist does not include sample and diluent volumes, enter the final concentration for the samples. This value is used with the final volume and the initial concentration to calculate how much sample and diluent to pipette for each sample.
10. Enter the final volume in µL for the normalized samples. If your worklist does not include sample and diluent volumes, this value is used with the initial and final concentrations to calculate how much sample and diluent to pipette for each sample.
    
    Whether the volumes are calculated or included in the worklist, this value is also the maximum amount of sample allowed. The max sample volume is used with the settings in the Error Handling tab to configure how the Prep will behave if a sample’s final concentration is too low.
11. Enter a minimum allowable sample volume in µL. This value is used with the settings in the Error Handling tab to configure how the Prep will behave if a sample’s concentration is too high.
12. Check the Error Handling settings. These settings control how the Prep responds when a normalized sample’s concentration is too high or too low.
    • If your worklist does not include sample and diluent volumes, then the handling for sample concentration errors can be set to one of two options. If a sample volume is below the minimum value from step 11 (resulting in a final concentration that is too high), you can choose to skip the sample or just pipette the minimum sample volume. If a sample volume is above the maximum value from step 10 (resulting in a final concentration that is too low), you can skip the sample or pipette the maximum sample volume.
      
      If your worklist does include the sample and diluent volumes, both settings default to skipping samples whose volume is too high or low.
    • If your worklist includes sample and diluent volumes, you can choose when to show errors caused by a mismatch between the final volume and the total sample and diluent volumes.
    • The Prep will automatically handle these errors as long as the calculated number of errors is below the number of “Maximum errors allowed”. If more errors will occur, an error dialog will appear that requires a user to choose whether to continue the run.
13. Tap Next after entering the Normalization settings.
14. Create a new diluent, or select an existing one:
    1. Tap New Diluent, or select it from the diluent drop-down menu. A dialog to create the diluent appears.
       
       To use an existing diluent or reagent from another step, select it from the drop-down menu.
    2. Enter a name for the diluent, select its liquid type, then tap Create. If none of the liquid types are similar enough to your diluent, learn about managing unknown liquids.
    3. Select the labware and containers that will hold diluent. These containers will be treated as a single source: the channels will aspirate repeatedly from one container until it is empty, then move on to the next. Tap Next when finished.
15. Enter the required low- and mid-volume pipetting settings for the diluent and samples. A tip type is required for each page, and the samples pages also require a liquid type.
16. Adjust the default settings in the Tip, Aspirate, Dispense, and Liquid tabs for each settings page (optional).
17. Tap Finish to complete the step. Verify the protocol and check for errors. All changes are saved automatically, so you can exit the protocol without losing progress after finishing the step.