How do I do volume verification on the Microlab Prep?

If your lab requires verification of the Microlab Prep’s liquid handling, built-in protocols are available to do so. High- and low-volume protocols are available for both the 2 Independent Channels and 8-Probe Head. The protocols require specific accessories, tips, and liquids to verify the Prep’s full pipetting range; double-check the Requirements section to make sure you have everything you need to perform verification.

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

Requirements

Available from Hamilton

Hamilton verification kit components: colored verification solutions, pipette tip racks, microplate strips, and black plate holders on white surface
  • 2 framed tip racks (not included with refill kit)
  • Blister of 1000 µL tips
    • Low-volume channel verification: 6 tips
    • High-volume channel verification: 20 tips
    • Low-volume 8 MPH verification: 24 tips
    • High-volume 8 MPH verification: 80 tips
  • Blister of 50 µL tips
    • Low-volume channel verification: 20 tips
    • Low-volume 8 MPH verification: 80 tips
  • 4 microplates (Nunc, 96, Solid, Flat Bottom, 400 µL, Clear, Polystyrene, Pinchbar)
  • 3 reagent reservoirs (Seahorse-Agilent, 1 cavity, 96 Btm, 300 mL, Clear PP)
  • Verification solutions 1–3

Required from Your Lab

  • Absorbance reader, single mode with 405nm filter OR dual mode with 405nm and 620-650 filter.
  • Balance with 1 mg resolution and accuracy

Procedure

Preparation

  1. Double-check that you have the required materials.

  2. Record the part and serial numbers of the pipetting tools you are verifying:

    a. Space out the pipetting tools as shown.

Hamilton automated liquid handler with MicroLab label; blue circle highlights a component on the rear channel

b. Record the part and serial numbers shown on the indicated stickers. The serial number is six digits long, with a three-digit dash number (XXXXXX–XXX). The stickers for the front channel and 8 MPH are easiest to see from the angle shown, while the rear channel’s sticker is visible from the front.

Close-up of a laboratory liquid handler's front channel area with two circled components highlighted
Three close-up photos showing barcode label stickers on liquid handling instrument channels, highlighted with blue rectangles.

3. Turn on the Prep if it is not already on. Sign in if required.

4. Open the Verification page. From the home page, tap the settings button in the top-right corner, open the Instrument tab, and tap Verification.

5. Open the spreadsheet for the pipetting tools you are verifying on a separate computer. Record the serial number(s) of the channels or 8 MPH, as well as the density listed for Solution 1 on the Verification Solutions’ Declaration of Quality.

6. Open the tab for the pipetting tool(s) you are verifying. The steps for the channel and 8 MPH protocols differ.

Low-Volume Channel Verification

Make sure to allocate enough time to perform each verification protocol uninterrupted. Delays between steps can significantly affect results.

  1. Tap the Low Volume Verification for Independent Channels protocol.
  2. Prepare the following consumables:
    • Reagent reservoir with half bottle of Solution 1 (unless already added)
    • Reagent reservoir with half bottle of Solution 2 (unless already added)
    • 50 µL conductive tips, no filter
    • 1000 µL conductive tips, no filter
    • Clean microplate
  3. If using a single mode reader, use a 405nm filter to measure the empty microplate. Record results in the "LV OD Calc" tab of the spreadsheet in the "Reference OD Results" table.
  4. Load the labware and confirm the deck layout using the camera, if available. Address any warnings, then start the protocol.
  5. Record the density of Solution 1 in the spreadsheet, if you have not yet done so. Continue the run when ready.

    The Prep will pipette 100 µL of Solution 2 to each well from A1 through B10.
  6. When the run pauses, carefully remove the microplate and tare the balance with the plate.
  7. Return the plate to site 8 and resume the run. The Prep will pipette 10 µL of Solution 1 to each well from A1 through B10.
  8. When the run pauses again, carefully remove the microplate and weigh it on the balance.
  9. Record the mass in the spreadsheet.
  10. Return the plate to site 8 and resume the run. The Prep will pipette 150 µL of Solution 2 to each well from A1 through B10.
  11. When the run pauses again, carefully remove the microplate. Resume the run to finish.
  12. Shake the plate for 40 s at high intensity.
  13. Place the plate in your lab’s absorbance reader and measure the plate:
     
    1. Dual Mode Reader
      1. Use a 405nm and 620-650nm filter to measure the microplate with solutions 1 and 2.
      2. If the reader auto calculates the difference between both readings, then record outputted ODs into the highlighted cells, 1A-10B, of the “Low Volume Photometric Results” table of the spreadsheet. Paste Values Only.
      3. If the reader outputs both readings, then record outputted ODs into the calculators on the “LV OD Calc” tab. Paste Values Only.
        1. Record 620-650nm OD values into the “Reference OD Results” Table
        2. Record 405nm OD values into the “Measurement OD Results” Table.
        3. Copy the Adjusted OD values into the highlighted cells, 1A-10B, of the “Low Volume Photometric Results” table of the spreadsheet. Paste Values Only.
    2. Reader without dual mode capability
      1. Use a 405nm filter to measure the microplate with solutions 1 and 2.
      2. Within the “LV OD Calc” tab of the spreadsheet, record the results in the “Measurement OD Results” table. The “Adjusted OD Results” table will be automatically populated.
      3. Copy the Adjusted OD values into the highlighted cells, 1A-10B, of the “Low Volume Photometric Results” table of the spreadsheet. Paste Values Only.
  14. The spreadsheet will display the result of the low-volume verification in Low Volume Results. Contact Hamilton if your Prep’s channels did not pass the criteria.

High-Volume Channel Verification

Make sure to allocate enough time to perform each verification protocol uninterrupted. Delays between steps can significantly affect results.

  1. Tare the balance with an empty microplate.
  2. In the Verification page, tap the High Volume Verification for Independent Channels protocol.
  3. Prepare the following consumables:
    • Reagent reservoir with half bottle of Solution 3 (unless already added)
    • 1000 µL conductive tips, no filter
    • Clean microplate
  4. If using a single mode reader, use a 405nm filter to measure the empty microplate. Record results in the "LV OD Calc" tab of the spreadsheet in the "Reference OD Results" table.
  5. Load the labware and confirm the deck layout using the camera, if available. Address any warnings, then start the protocol.

    The Prep will pipette 250 µL of Solution 3 to each well from A1 through B10.
  6. When the run pauses, carefully remove the microplate and weigh it on the balance.
  7. Record the mass in the spreadsheet.
  8. Shake the plate for 10 s at high intensity.
  9. Place the plate in your lab’ absorbance reader and measure the plate:
     
    • Dual Mode Reader
      • Use a 405nm and 620-650nm filter to measure the microplate with solutions 1 and 2.
      • If the reader auto calculates the difference between both readings, then record outputted ODs into the highlighted cells, 1A-10B, of the “Low Volume Photometric Results” table of the spreadsheet. Paste Values Only.
      • If the reader outputs both readings, then record outputted ODs into the calculators on the “LV OD Calc” tab. Paste Values Only.
        • Record 620-650nm OD values into the “Reference OD Results” Table
        • Record 405nm OD values into the “Measurement OD Results” Table.
        • Copy the Adjusted OD values into the highlighted cells, 1A-10B, of the “Low Volume Photometric Results” table of the spreadsheet. Paste Values Only.
    • Reader without dual mode capability
      • Use a 405nm filter to measure the microplate with solutions 1 and 2.
      • Within the “LV OD Calc” tab of the spreadsheet, record the results in the “Measurement OD Results” table. The “Adjusted OD Results” table will be automatically populated.
      • Copy the Adjusted OD values into the highlighted cells, 1A-10B, of the “Low Volume Photometric Results” table of the spreadsheet. Paste Values Only.
  10. The spreadsheet will display the result of the high-volume verification in High Volume Results. Contact Hamilton if your Prep’s channels did not pass the criteria.

Low-Volume 8 MPH Verification

Make sure to allocate enough time to perform each verification protocol uninterrupted. Delays between steps can significantly affect results.

  1. Tap the Low Volume Verification for 8 MPH protocol.
  2. Prepare the following consumables:
    • Reagent reservoir with half bottle of Solution 1 (unless already added)
    • Reagent reservoir with half bottle of Solution 2 (unless already added)
    • 50 µL conductive tips, no filter
    • 1000 µL conductive tips, no filter
    • Clean microplate
  3. If using a single mode reader, use a 405nm filter to measure the empty microplate. Record results in the "LV OD Calc" tab of the spreadsheet in the "Reference OD Results" table.
  4. Load the labware and confirm the deck layout using the camera, if available. Address any warnings, then start the protocol.
  5. Record the density of Solution 1 in the spreadsheet, if you have not yet done so. Continue the run when ready.

    The Prep will pipette 100 µL of Solution 2 to each well from A1 through H10.
  6. When the run pauses, carefully remove the microplate and tare the balance with the plate.
  7. Return the plate to site 8 and resume the run. The Prep will pipette 10 µL of Solution 1 to each well from A1 through B10.
  8. When the run pauses again, carefully remove the microplate and weigh it on the balance.
  9. Record the mass in the spreadsheet.
  10. Return the plate to site 8 and resume the run.The Prep will pipette 150 µL of Solution 2 to each well from A1 through H10.
  11. When the run pauses again, carefully remove the microplate. Resume the run to finish.
  12. Shake the plate for 40 s at high intensity.
  13. Place the plate in your lab’s absorbance reader and measure the plate:
     
    • Dual Mode Reader
      • Use a 405nm and 620-650nm filter to measure the microplate with solutions 1 and 2.
      • If the reader auto calculates the difference between both readings, then record outputted ODs into the highlighted cells, 1A-10B, of the “Low Volume Photometric Results” table of the spreadsheet. Paste Values Only.
      • If the reader outputs both readings, then record outputted ODs into the calculators on the “LV OD Calc” tab. Paste Values Only.
        • Record 620-650nm OD values into the “Reference OD Results” Table
        • Record 405nm OD values into the “Measurement OD Results” Table.
        • Copy the Adjusted OD values into the highlighted cells, 1A-10B, of the “Low Volume Photometric Results” table of the spreadsheet. Paste Values Only.
    • Reader without dual mode capability
      • Use a 405nm filter to measure the microplate with solutions 1 and 2.
      • Within the “LV OD Calc” tab of the spreadsheet, record the results in the “Measurement OD Results” table. The “Adjusted OD Results” table will be automatically populated.
      • Copy the Adjusted OD values into the highlighted cells, 1A-10B, of the “Low Volume Photometric Results” table of the spreadsheet. Paste Values Only.
  14. The spreadsheet will display the result of the low-volume verification in Low Volume Results. Contact Hamilton if your Prep’s 8 MPH did not pass the criteria.

High-Volume 8 MPH Verification

Make sure to allocate enough time to perform each verification protocol uninterrupted. Delays between steps can significantly affect results.

  1. Tare the balance with an empty microplate.
  2. In the Verification page, tap the High Volume Verification for 8 MPH protocol.
  3. Prepare the following consumables:
    • Reagent reservoir with half bottle of Solution 3 (unless already added)
    • 1000 µL conductive tips, no filter
    • Clean microplate
  4. If using a single mode reader, use a 405nm filter to measure the empty microplate. Record results in the "LV OD Calc" tab of the spreadsheet in the "Reference OD Results" table.
  5. Load the labware and confirm the deck layout using the camera, if available. Address any warnings, then start the protocol.

    The Prep will pipette 250 µL of Solution 3 to each well from A1 through H10.
  6. When the run pauses, carefully remove the microplate and weigh it on the balance.
  7. Record the mass in the spreadsheet.
  8. Shake the plate for 10 s at high intensity.
  9. Place the plate in your lab's absorbance reader and measure the plate:
     
    • Dual Mode Reader
      • Use a 405nm and 620-650nm filter to measure the microplate with solutions 1 and 2.
      • If the reader auto calculates the difference between both readings, then record outputted ODs into the highlighted cells, 1A-10B, of the “Low Volume Photometric Results” table of the spreadsheet. Paste Values Only.
      • If the reader outputs both readings, then record outputted ODs into the calculators on the “LV OD Calc” tab. Paste Values Only.
        • Record 620-650nm OD values into the “Reference OD Results” Table
        • Record 405nm OD values into the “Measurement OD Results” Table.
        • Copy the Adjusted OD values into the highlighted cells, 1A-10B, of the “Low Volume Photometric Results” table of the spreadsheet. Paste Values Only.
    • Reader without dual mode capability
      • Use a 405nm filter to measure the microplate with solutions 1 and 2.
      • Within the “LV OD Calc” tab of the spreadsheet, record the results in the “Measurement OD Results” table. The “Adjusted OD Results” table will be automatically populated.
      • Copy the Adjusted OD values into the highlighted cells, 1A-10B, of the “Low Volume Photometric Results” table of the spreadsheet. Paste Values Only.
  10. The spreadsheet will display the result of the high-volume verification in High Volume Results. Contact Hamilton if your Prep’s 8 MPH did not pass the criteria.