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Gravimetric Method
 



According to ISO 8655, for each volume of interest

 

 

  1. Record the temperature, humidity and pressure. Ensure that these environmental conditions meet the controlled requirements.

  2. Place a dry, clean microtiter plate onto balance and tare the balance, which will give zero reading on the display.

  3. Pipette a desired volume of the distilled water using the robot under test.

  4. Transport the microtiter plate with dispensed liquid and place it gently onto the balance.

  5. Allow the display to stabilize and record the mass , retrieve the microtiter plate and then tare the mass again.

  6. Repeat the Step 3 through 6 till five measurements have been recorded. It is suggested to use new tips every cycle.

  7. Estimate the evaporation loss

  8. Repeat Step 1. Check the environmental conditions are still within requirements.

Single-Dye Fluorescence-Based Method



The Florescence-Based Method was used for volume of interest that was larger than 50 μL.

 

  1. Prepare the Fluorescein solution.

  2. Generation of the standard curve: dispense a series of target volumes of the Fluorescein solution into a black fluorescence-specific microtiter plate with a well-calibrated pipette. Then measure the fluorescence intensity of these solutions and plot volume versus fluorescence intensity so as to obtain a regression line. The coefficient of determination of the linear curve must be larger than 0.99. Otherwise, this step should be repeated.

  3. Place a reservoir that contains sufficient Fluorescein solution and another clean microtiter plate specified for fluorescence spectroscopy on the deck of the liquid handling robot.

  4. Set a target volume (> 50 μL) for liquid transfer. Using clean and appropriate tips, cycle the liquid handling robot under test through a full method.

  5. Check if there are bubbles in the wells. If so, remove the bubbles by aspirating and dispensing with a pipette carefully.

  6. Measure the fluorescence intensity of each well by the microplate reader.

  7. Repeat the Step 4 through 6 till three measurements are recorded. It is suggested to use new tips every cycle.

  8. Import data into an Excel template for rapid data processing.

Dual-Dye Absorbance-
Based Method



For liquid volume below 50 μL, the Absorbance-Based Method was performed.

 

  1. Prepare the sample solution and the diluent.

  2. Determination of the molar absorptivities

  3. Place the labware on the deck of the liquid handling robot, including two reservoirs contain sample solution and diluent respectively and a clean microtiter plate specified for absorbance measurement.

  4. Set a target volume for liquid transfer. Using clean and appropriate tips, cycle the liquid handling robot under test through a full method.

  5. Add diluent to each well with the same liquid handling robot pod to the recommended total volume of approximately 100 μL to allow meaningful absorbance readout.

  6. Centrifuge the microtiter plate for 10 minutes in the plate reader for mixing.

  7. Measure the dual-dye absorbance of each well in the microtiter plate at two wavelengths by the microplate reader.

  8. Open the door of the plate reader and check if there are bubbles in the well. If so, repeat Step 6 and 7.

  9. Repeat the Step 4 through 8 till three measurements are recorded. It is suggested to use new tips every cycle.

  10. Baseline measurement: measure the absorbance of 100 μL diluent at wavelength 450 nm and the absorbance of 100 μL distilled water at wavelength 600 nm, respectively. Each liquid is dispensed in the same procedures as Step 4 through Step 8.

  11. Import data into an Excel template for rapid data processing.

EXPERIMENT

 

On the basis of the theories described in the Theory Page, three volume verification methods are adapted according to the availability of the materials and devices in the Biolab. 

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