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Art & Science of
Liquid Handling



Robotic Sample Processors

tubing. The Liquid Class Database allows the management of all factors that influence liquid handling in an organized manner. conductivity). Airgaps and recommended settings for water Example Airgap Software Definition Settings System Trailing Airgap (STAG) Airgap after system fluid Default range: 4-10 µl Leading Airgap (LAG) Airgaps before aspirated liquid volume Default range: 1-10 µl Trailing Airgap (TAG) Airgap after last aspirated Default range: liquid volume. viscosity. double or no (off) detection. The Liquid Class Database will not only save you time when developing your first robot application. Z-Values Liquid Class Database Tecan’s Gemini software for applications development incorporates a unique new tool to help developers of robotic applications manage these factors.e. Pipetted liquids can have both leading and trailing airgaps. Each of these factors affect the accuracy and precision of liquid handling. and DMSO. Z-values and recommended settings Example ➝ 0 Z-Value Definition Setting Z-Travel Height at which the tip travels between positions within a rack Set higher than any possible obstruction Z-Start Height at which the MiniPrep activates Liquid Detection Operation Set just inside the neck of the vessel and just above the liquid Z-Dispense Height in the vessel at which the MiniPrep dispenses liquid Set based on your needs. • Wash Procedures • Additional Precision and Accuracy Considerations • Default Settings and Procedures Pipetting Modes Table 1 defines three different modes. must be above the liquid level Z-Max Lowest height in the vessel in the Z-axis For routine operation. but it will allow you to apply your experience quickly to subsequent methods that you may develop. Z-values represent the vertical heights set for the pipette tip as it moves. Choices include single. and dispenses. Liquid classes include water. Airgaps Airgaps separate different liquids in the pipetting tips (separation airgap) and tubing and prevent droplets at the end of the tip while the arm moves over the worktable (trailing airgap). serum. Pre-programmed liquid classes are provided. then edit the default settings to create new custom liquid classes. set Z-max to 5 steps above the bottom of the vessel steps ➝ ~1600 . characteristics of the fluid itself are important (i. volume. ethanol). including disposable tips (DiTi). rename. These include pump mechanics. Pipetting modes Single Pipetting Mode Definition Single A single probe aspirates the liquid volume (one aliquot) at one source position and dispenses the entire volume at one destination position Dual A dual probe aspirates and dispenses the liquid volume (one aliquot) through each barrel Multi A single or dual probe aspirates and dispenses multiple liquids (multiple aliquots) multiple times System Fluid STAG LAG Vol. Table 3. In addition. Double detection was used for this technical note. Refer to the MiniPrep manual for additional information. This note provides fundamental concepts that will help you optimize your automated liquid handling applications and includes the following topics: • • • • • Pipetting Modes Z-Values Liquid Level Detection Airgaps Aspiration and Dispense Speeds Multi-pipetting includes additional parameters than will be discussed here. Table 2.g. Other tips are available. aspirate and dispense speeds. The user can copy.Many factors come into play when aspirating and dispensing fluids. alcohol (e. prevents 1-5 µl droplet formation at end of the tip Table 1. Single pipetting was used to obtain data presented in this note. Liquid detection is crucial for the instrument to ensure aspiration of the liquid and minimal carry over of liquid on the outer surfaces of the tip. and the airgaps that separate the sample from the system fluid. Table 2 describes each Z-value. aspirates. and dispensed together at the same destination. Airgaps are treated together with their associated liquids. Liquid Level Detection Liquid detection mode defines the manner in which the MiniPrep attempts to detect liquid in the container.

The waste line aspirates the requested volume from the last tube and dispenses it to the waste position. • Small volumes Relatively higher dispense speeds are required for volumes < 20 µl. Typically. Please refer to the MiniPrep manual for more information. When optimizing the dispense speed. speed. Low speeds are needed when pipetting small volumes (<25 µl). Several small washes are more efficient than a single large one. Additional Precision and Accuracy Considerations typical range is 100600 µl/s In general. set the minimum wash volume to 5 ml. This activates the pump for 1 to 2 seconds. After about 12 aspirate/dispense movements with a DiTi. During liquid aspiration a vacuum builds inside the system tubing. • Serial dilutions are prepared in destination racks. One mixing cycle is best. • Large volumes Larger volumes lose accuracy when dispensed with high speeds due to dilution effects and breaking of airgaps. During the operation. a default 50% of the volume dispensed to a serial or pre-dilution well is re-aspirated and dispensed. dispense faster. insert a waste line at the end of the dilution series. a 10 µl sample requires a 1000 µl syringe or smaller. Table 4.06 inch tubing).Aspiration and Dispense Speeds Wash Procedures Liquid handling techniques employ the general rule: aspirate slow. for syringe < 1 ml use smaller inner diameter tubing. at fixed settings. a decrease of aspiration speed will increase precision and accuracy. Larger volumes should be dispensed at relatively slower speeds. When foaming is present. CAUTION: Retracting the tip from the liquid too quickly can result in aspiration of air instead of liquid. For pipetted liquid volume <30 µl/s. you don’t need to wash. accuracy requirement. The MiniPrep has speeds ranging from 1. Using deionized water for diluting samples or reagents may interfere with liquid detection. pushes liquid through the tip at the flow rate of ~180 ml per minute. or can introduce bubbles into the system. You can set the breakoff from 0-24. It ensures the plunger moves to the correct position. • Foaming and droplet formation Liquids such as detergents must be dispensed slowly to avoid foaming and droplet formation on the outside of the tip. default = 0 ms When optimizing liquid handling procedure: • wash volume must be sufficient to empty the clean station • wash speed must be set to create turbulence to clean the outside of the tip In general use a larger wash volume whenever possible. Aspiration settings for water Aspirate Dispense Definition Settings Speed: the flow rate at the tip when aspirating the liquid volume Delay: a pause in the syringe movement after aspirating the liquid volume. wash the tips between pipetting steps since this will aspirate a new STAG. and breakoff. due to minimization of active ions. and provides fast and effective washing to enable faster processing times. To ensure a constant volume of liquid for all dilution steps. Rapid aspiration speeds can cause the airgap to break up which results in a significant dilution effect. Viscosity of the liquid. The faster the liquid is aspirated. Faster dispense speeds generally result in better break off at the tip. Other factors to consider are the smallest and largest volumes pipetted. • System Liquid should always be deionized water • The MiniPrep’s digital pump uses a stepper motor to drive the plunger. including the one at the end of the sequence. the STAG is gone. gives the system time to return to equilibrium typical range is 10150 µl/s Speed: the flow rate at the tip when dispensing the liquid volume 100 ms is sufficient for most applications. Routine pipetting with steel tips requires both wash and flush steps to ensure proper cleaning of the tips between pipetted liquids. Specifying a maximum mix volume minimizes carry over. then a larger syringe will allow more aliquots. When using the Miniwash. • Use Tracking to avoid dispensing directly into the liquid or to avoid foaming while dispensing. Backlash compensation is built into the firmware. Below is a brief description of other parameters.2 seconds per stroke to 20 minutes per stroke. >1000 µl is suggested. which is critical to liquid detection. not standard 0. tubing size. NOTE: For the best precision and accuracy. the tip submerges a set number of steps in the liquid. syringe. When using a DiTi. • Syringe Selection: generally small syringes maximize accuracy and precision. A Miniwash Pump (an option). • The Breakoff indicates the number of steps not performed at the end of the plunger strokes. . The aspiration delay gives the system time to return to equilibrium. liquids with high viscosity or low boiling points. a high velocity membrane pump. • After detecting liquid. In normal liquid. the stronger this vacuum pulls. Viscous liquids require an appropriate aspiration delay. Use flush before starting a procedure as a means of priming and cleaning the system. and tip configuration may effect speeds. called Backlash. NOTE: Dispensing with tracking overrules the Z-dispense setting. The choice of aspiration and dispense speeds affects both accuracy and precision. for pipetted liquid volume >50 µl use 2500 µl. TAG). A mechanical tolerance. • The type of liquid Different viscosities and compositions require different liquid handling parameters. Match syringe size with appropriate tubing (e. the syringe size should not exceed 100 times the smallest sample volume. set the breakoff to 10 (the default). use double detection and increase the submerge steps between 10 and 25 steps. consider the following when dispensing from air: • Calculation of the total volume to be dispensed This total includes all volumes in a pipetting sequence including the airgaps (LAG. For mixing.g. set the Submerge Steps to 5. • Pre-Dilutions are prepared in separate racks. For example. exists between the gears in the drive train. If the application requires multiple aspirations or multiple dispenses. but flush the system between use to ensure hydraulic operation. Meiji Seimei Building 10F. Water liquid class screen example from software. Singapore 459 231. single pipetting Coefficient of variation: CV % = (stdev / average) *100 2 Accuracy %: standard • Tecan U. 5082 Grödig/ Salzburg. every single distribution step should be repeated at least 15 times.. standard XL3000.0% <5% <1. RG8 9AR. A matrix composed of three XL 3000 diluters. Volta 16. 1-40. Fuchu City. Within each volume range at least three volumes were measured. Italy. E-mail: info@tecan. Calculate average. Tokyo 183. Calibration example. Keep in mind to correct the volume by the density of the liquid. CA 95131. Fax: +49 7951 5038. Austria. Box 13953. Tel: +33 1 30 68 81 50. Research Triangle Park. 1 ml syringe on a standard XL 3000 diluter with 3 port valve. 6. Fax: +1 919 361 5201.. Fax: +44 1491 875432. and water pipetted. Table 5. Tel: +39 2 267 00553. E-mail: tecanuk@compuserve. 2450 Zanker Road. Fax: +33 1 30 68 98 13 • Tecan Italia S.r. Parc d’Activités de Tel: +65 4441 886. Tel: +44 1491 875087. 3.HERMES II. San Jose. Run the model process in single step • Tecan Asia (Pte) Ltd. Measured Volume (µL) 100 80 60 40 20 0 0 20 40 60 80 100 120 Expected Volume (µL) • Tecan AG. E-mail: tecan@tecan. 3 port valve 120 Set value – Actual value *100 Set value Gravimetric Calibration Procedure: 1. Values from your balance can be collected with a special data acquisition software. E-mail: • Cavro Scientific Instruments Inc. Tel: +41 55 254 81 11.O.. Via A. Ltd. Feldbachstrasse 80. Hotline: 800 338 3226. Only 1 tip can be used at a time. USA. • Tecan UK Ltd. 20093 Cologno Monzese (MI). USA. low. Tel: +1 408 953 3100. Tel: +43 6246 8933. Bâtiment B .de • Tecan France SA. 18 The High Street. Fax: +1 408 953 3107. E-mail: tecan@tecan. 2.. Fax: +41 55 244 38 83. Rue Edouard Branly. Fax: +65 4441 836. _ Standard deviation (Stdev): s = 1 Σ(x – x) 2 Figure 2. E-mail: cavro@cavro. Japan. In the instrument software define your balance (Mettler AT261 or equivalent) as a destination rack. Untersbergstrasse 1a. Fax: +43 6246 72770. Germany. Use the Application Software to compose a model process (method) for each liquid transfer using the same volumes and liquid handling parameters. Precision and accuracy for standard tip* Volume dispensed from air (µl) Precision1 (CV) Accuracy2 +/- 10 100 < 3% < • Tecan Japan Co. Theodor Storm-Strasse 17. precision (CV) and accuracy of your data. France. The Worktable should also include source (tube racks) and wash station. Tel: +81 4 2334 8855. E-mail: tecan@tecan. An example of a linear regression line is seen in Figure 2. Tel: +1 919 361 5200. Miyamachi. 74564 Crailsheim.. Goring-on-Thames. three syringes and three standard tips was used (20 dispenses per combination of diluter-syringe-standard tip). 26 East Coast Avenue. NC 27709. Reading. • Tecan Austria GmbH. Fax: +39 2 253 2890.0% * Each volume was dispensed 540 times from air. Determine a calibration curve. 78190 Trappes. Fax: +81 4 2334 0401. Description of gravimetric procedure is given below (a photometric procedure is available too).Figure 1.l. After each dispense read the actual mass from the balance display. medium and high. E-mail: Windows is a trademark of Microsoft Corporation P/N 730317-002 Printed in the USA 4/00 . Tel: +49 7951 94170. Inc. 4. n–1 Water Liquid Class. 5. E-mail: • Tecan Deutschland GmbH. Hotline: +41 55 254 82 82. For statistical reasons. CH-8634 Hombrechtikon. Default Settings Specifications on precision and accuracy for the MiniPrep are based on gravimetric measurements of a single pipetting process using a standard tip. 1mL P.