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How do I do volume verification?

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

  • Volume verification spreadsheet(s)
  • Volume verification kit (order a kit with racks or a refill kit)
    • 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 with 405 nm filter and 620–650 nm 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:
    1. Space out the pipetting tools as shown.

    2. 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.


  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. Load the labware and confirm the deck layout using the camera, if available. Address any warnings, then start the protocol.
  4. 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.
  5. When the run pauses, carefully remove the microplate and tare the balance with the plate.
  6. 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.
  7. When the run pauses again, carefully remove the microplate and weigh it on the balance.
  8. Record the mass in the spreadsheet.
  9. 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.
  10. When the run pauses again, carefully remove the microplate. Resume the run to finish.
  11. Shake the plate for 40 s at high intensity.
  12. Place the plate in your lab’s absorbance reader and measure the plate:
    • Dual-mode readers:
      Measure the plate using a 405 nm filter and a 620–650 nm filter, then record the measured ODs in the highlighted cells (A1–B10) of the “Low Volume Photometric Results” table in the spreadsheet. Copy and paste the values to avoid errors.
    • Single-mode readers:
      Perform two measurements: one with a 405 nm filter, and one with a 620–650 nm filter. Record each set of measurements in the “LV OD Calc” tab of the spreadsheet in the corresponding tables (“Low Volume 405 nm Results” and “Low Volume 620–650 nm Results”).

      Copy the auto-calculated “Low Volume Adjusted OD” values into the “Low Volume Photometric Results” table. Copy and paste the values to avoid errors.
  13. 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. 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.
  5. When the run pauses, carefully remove the microplate and weigh it on the balance.
  6. Record the mass in the spreadsheet.
  7. Shake the plate for 10 s at high intensity.
  8. Place the plate in your lab’ absorbance reader and measure the plate:
    • Dual-mode readers:
      Measure the plate using a 405 nm filter and a 620–650 nm filter, then record the measured ODs in the highlighted cells (A1–B10) of the “High Volume Photometric Results” table in the spreadsheet. Copy and paste the values to avoid errors.
    • Single-mode readers:
      Perform two measurements: one with a 405 nm filter, and one with a 620–650 nm filter. Record each set of measurements in the “HV OD Calc” tab of the spreadsheet in the corresponding tables (“High Volume 405 nm Results” and “High Volume 620–650 nm Results”).

      Copy the auto-calculated “High Volume Adjusted OD” values into the “High Volume Photometric Results” table. Copy and paste the values to avoid errors.
  9. 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. Load the labware and confirm the deck layout using the camera, if available. Address any warnings, then start the protocol.
  4. 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.
  5. When the run pauses, carefully remove the microplate and tare the balance with the plate.
  6. 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.
  7. When the run pauses again, carefully remove the microplate and weigh it on the balance.
  8. Record the mass in the spreadsheet.
  9. 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.
  10. When the run pauses again, carefully remove the microplate. Resume the run to finish.
  11. Shake the plate for 40 s at high intensity.
  12. Place the plate in your lab’s absorbance reader and measure the plate:
    • Dual-mode readers:
      Measure the plate using a 405 nm filter and a 620–650 nm filter, then record the measured ODs in the highlighted cells (A1–B10) of the “Low Volume Photometric Result” table in the spreadsheet. Copy and paste the values to avoid errors.
    • Single-mode readers:
      Perform two measurements: one with a 405 nm filter, and one with a 620–650 nm filter. Record each set of measurements in the “LV OD Calc” tab of the spreadsheet in the corresponding tables (“Low Volume 405 nm Results” and “Low Volume 620–650 nm Results”).

      Copy the auto-calculated “Low Volume Adjusted OD” values into the “Low Volume Photometric Results” table. Copy and paste the values to avoid errors.
  13. 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. 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.
  5. When the run pauses, carefully remove the microplate and weigh it on the balance.
  6. Record the mass in the spreadsheet.
  7. Shake the plate for 10 s at high intensity.
  8. Place the plate in your lab's absorbance reader and measure the plate:
    • Dual-mode readers:
      Measure the plate using a 405 nm filter and a 620–650 nm filter, then record the measured ODs in the highlighted cells (A1–B10) of the “High Volume Photometric Results” table in the spreadsheet. Copy and paste the values to avoid errors.
    • Single-mode readers:
      Perform two measurements: one with a 405 nm filter, and one with a 620–650 nm filter. Record each set of measurements in the &ldquo:HV OD Calc” tab of the spreadsheet in the corresponding tables (“High Volume 405 nm Results” and “High Volume 620–650 nm Results”).

      Copy the auto-calculated “High Volume Adjusted OD” values into the “High Volume Photometric Results” table. Copy and paste the values to avoid errors.
  9. 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.

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