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96 Well Pipetting Precision and Accuracy Using the RapidPlate 96/384 Workstation

Lynn Jordan1 and Brent Butler2
1Caliper Life Sciences Inc., Applications Laboratory, Hopkinton, Massachusetts, USA
2GlaxoSmithKline, Department of Molecular Screening, Research Triangle Park, North Carolina, USA

Introduction

Initially, work in 96 well microplates was commonly carried out using manual pipetting. As the demand for greater throughput of plate processing has grown, automated liquid handling has become a necessity for most laboratories. The most critical aspect of an automated liquid handler is to understand its performance, typically determined by precision and accuracy.

At Caliper Life Sciences, precision and accuracy testing is achieved by dispensing a tartrazine solution into microplates containing water. This is referred to as a "liquid-to-liquid" dispense. The purpose of this paper is to report the performance obtained by dispensing 0.5 to 2μL aqueous samples containing tartrazine into 96 well plates containing water. Precision and accuracy results are shown for this work.

Materials and Methods

Instrumentation
Testing was performed using a RS-232 RapidPlate 96/384 Workstation (Caliper Life Sciences) using 100μL Automation Certified Pipette TipsTM (Caliper Life Sciences). A Bulk Dispenser (Caliper Life Sciences) was utilized for bulk dispensing across the columns of the microplate (secondary dispensing). Absorbance was measured at 450nm using a Wallac Victor2 Multilabel Counter (PerkinElmer).

Reagents
Tartrazine (Sigma Chemical Company, Catalogue number T-0388) is dissolved in deionized water. Concentrations of 6.0, 1.4, 0.7 and 0.14g/L corresponding to both 1 and 2, 5, 10 and 25μL dispenses respectively are prepared by dissolving the tartrazine into 1L of deionized water. The tartrazine solution was dispensed into plates containing a volume of degassed deionized water. The deionized water was degassed by bubbling helium through the water for a period of 15 minutes.

Qualification of a Reagent Dispenser
Qualification of a bulk reagent dispenser is done to confirm the accuracy of the volume dispensed. When testing the RapidPlate for low volume dispensing, 100μL of degassed water is added to the plates both before and after the tartrazine dispense. This is done on an independent dispenser. This bulk dispenser is qualified by dispensing volumes of water into a plate, which is then weighed on a balance, and the accuracy calculated. For our work, a Bulk Reagent Dispenser on a Sciclone Liquid Handler (Caliper Life Sciences) was used.

Calibration of the RapidPlate
The RapidPlate is calibrated for accuracy prior to precision testing (see, Jordan L and Cunningham J "Calibration of Dispensing Accuracy in the RapidPlate, Application Note, Caliper Life Sciences Inc., in press 2001). A gravimetric confirmation of the dispensed volumes is repeated to verify the accuracy of the calibrated dispensed volumes.

Setting up the RapidPlate
In the RapidPlate software the "Procedure Editor" is selected. Next, using the "Setup Tab" the deck layout is created with a reservoir, a tip box and one White 96 Well Clear-bottom un-treated plate (COSTAR), See Figure 1. The reservoir is filled with the tartrazine dispensing solution appropriate for the volume to be tested. The level of the liquid in the reservoir should be such that the tips just break the surface of the solution. The 96 Well plate contains 100μL of degassed water, dispensed by a qualified liquid handler. A clean box of 100μL pipette tips are used for every dispense.

Running the RapidPlate Procedure
For the Liquid-to-liquid dispense a RapidPlate procedure was written to attach tips, aspirate a volume of tartrazine solution from the reservoir, and then dispense into a clear bottom plate containing 100μL of degassed water. The procedure was designed to dispense into a single plate. Apart from appropriate changes in volumes, the same protocol was used for all volumes tested (Figure 2).

Mixing the Plates
After pipetting the 5 plates, 100μL of deionized water was added to each plate using a qualified reagent dispenser to aid in the mixing of liquids in the well. Next, the plates were read.

Reading the Microplates
Plates were read for absorbance at 450nm. The raw results were exported to ExcelTM and CV's were calculated for each volume.

Results

A gravimetric dispense test is done to determine the accuracy of the dispense operation after calibration of dispensing accuracy (Table 1).

Discussion

The experimental data shows that excellent pipetting performance can be obtained from the RapidPlate 96/384 Workstation. After calibration, we achieved accuracy levels of less than 1% error for volumes of 50, 20, 10, and 5μL, and errors of 2% and 30% for volumes of 2μL and 1μL respectively. While 30% error on the 1μL accuracy may give reason for concern, this can be easily reduced by entering additional gravimetric data points at these lower volumes when performing the calibration.

The dispense specifications for precision of the RapidPlate 96/384 Workstation are less than 5%CV for volumes of 1- 10μL, and 2% for volumes greater than 10μL, (for a liquid to liquid aqueous dispense). In our study, the CV's achieved are better than the specifications, especially taking into account actual volumes dispensed at 1 μL were significantly less, as the calibration was performed across the entire dispensing range, and not optimized for the lower volumes.


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