3 simple and proven automation protocols for serial dilutions on the ASSIST PLUS pipetting robot

  • 3 simple and proven automation protocols for serial dilutions on the ASSIST PLUS pipetting robot

    Serial dilution made easy

    Serial dilution – a reduction in concentration by a constant dilution factor – is a common approach for screening-related applications, such as determining minimum inhibitory concentrations (MIC) in drug discovery, calculating the most probable numbers (MPN) in microbiology, and performing general nucleic acid quantifications in molecular biology. Although it is a simple technique, poor liquid handling during interdependent dilution steps can cause error propagation and accumulation. Thorough mixing is therefore crucial, but this puts a lot of strain on the thumb, which increases the risk of repetitive strain injuries. In addition, performing serial dilutions regularly can be a time-consuming process.

    This application note describes the simplest way to do serial dilutions, using the VOYAGER adjustable tip spacing pipette on the ASSIST PLUS pipetting robot to gain more walk-away time. The protocols provided outline the optimal settings to ensure reliable results when diluting analytes in water. For further information about modifying key parameters to suit varying conditions, see INTEGRA's comprehensive guide to performing serial dilution.

  • Table of contents

    Serial dilution – a reduction in concentration by a constant dilution factor – is a common approach for screening-related applications, such as determining minimum inhibitory concentrations (MIC) in drug discovery, calculating the most probable numbers (MPN) in microbiology, and performing general nucleic acid quantifications in molecular biology. Although it is a simple technique, poor liquid handling during interdependent dilution steps can cause error propagation and accumulation. Thorough mixing is therefore crucial, but this puts a lot of strain on the thumb, which increases the risk of repetitive strain injuries. In addition, performing serial dilutions regularly can be a time-consuming process.

    This application note describes the simplest way to do serial dilutions, using the VOYAGER adjustable tip spacing pipette on the ASSIST PLUS pipetting robot to gain more walk-away time. The protocols provided outline the optimal settings to ensure reliable results when diluting analytes in water. For further information about modifying key parameters to suit varying conditions, see INTEGRA's comprehensive guide to performing serial dilution.

Key benefits

  • Proven serial dilution protocols, with optimal settings for the VOYAGER on the ASSIST PLUS, guarantee uniform pipetting and mixing.
  • The VOYAGER offers flexible automated serial dilutions across various tubes and plates, as well as the ability to switch pipette volumes seamlessly while maintaining the same protocol.
  • INTEGRA's electronic pipettes prevent thumb strain during liquid handling steps and, together with the ASSIST PLUS, enable risk-free handling of hazardous samples.
  • The ASSIST PLUS gives users additional hands-free time, eliminating time-consuming manual procedures.
  • These efficient liquid handling solutions support 2-, 5- and 10-fold serial dilutions, with dynamic mixing volumes ensuring homogeneity of analytes.
  • Simplified workflows are achieved with VIALAB's serial dilution protocol, which includes specific mixing parameters for managing poorly soluble analytes.
  • More flexibility during automated serial dilution with the VOYAGER adjustable tip spacing pipette when performing serial dilutions between different tubes and plates. Furthermore, the pipette can be easily changed to another volume size while using the same protocol.

Overview: How to do serial dilution with the ASSIST PLUS pipetting robot

This application note demonstrates how to perform serial dilution of tartrazine in water with an 8 channel 125 μl VOYAGER on the ASSIST PLUS.

Experimental set-up

The ASSIST PLUS, together with the 125 μl 8 channel VOYAGER and 125 μl sterile, filter GRIPTIPS® pipette tips, automates complete serial dilutions in 1 program consisting of 3 steps (Figure 1):

  1. Transfer diluent to target plate
  2. Transfer analyte to target plate
  3. Serial dilution of analyte within target plate
Illustration of a step-by-step procedure of how to do serial dilutions.
Figure 1: Experimental set-up for serial dilutions.

Downloads: App note and protocols for serial dilutions on the ASSIST PLUS pipetting robot

Step-by-step procedure

Graphical representation of the ASSIST PLUS pipetting robot deck set-up with INTEGRA dual adapter for reagent reservoirs, a 96 well flat-bottom plate and the VOYAGER adjustable tip spacing pipette.
Figure 2: Deck set-up for performing serial dilutions. Position A: Source – dual reservoir adapter with 2x10 ml reservoirs; diluent in A1 (blue) and analyte in A2 (green). Position B: Target – 96 well flat bottom plate (pink). Position C: Empty.

Serial dilution of an analyte

Serial dilution of an analyte

The INTEGRA dual reservoir adapter, together with 2x10 ml reservoirs, is placed on deck Position A, with diluent (blue) in A1 and analyte (green) in A2 (Figure 2). A clear 96 well flat bottom plate (pink) is placed in landscape orientation on deck Position B (Figure 2).

Select and run one of the following VIALAB programs:

2-fold serial dilution → 125_VOYAGER_2_fold_serial_dilution
5-fold serial dilution → 125_VOYAGER_5_fold_serial_dilution
10-fold serial dilution → 125_VOYAGER_10_fold_serial_dilution

Specific volumes are handled by the VOYAGER (Figure 3). The diluent is transferred in multiple dispensing steps from the reservoir (Position A – A1) into each well of the 96 well flat bottom plate, starting with column 2 (Position B). To ensure precision during plate set-up, a pre- and post-dispense of 5 % of the transferred volume is used for the 10-fold serial dilution, and 10 % for the 2- and 5-fold serial dilutions.

Illustration of 2-fold serial dilution, 5-fold serial dilution and 10 fold serial dilutions in 96 well flat bottom plates when using the 125 µl VOYAGER adjustable tip spacing pipette.
Figure 3: Serial dilutions in 96 well flat bottom plates with the 125 μl VOYAGER.

Using new GRIPTIPS, the VOYAGER aspirates the highest concentration of analyte from the reservoir (Position A – A2) and dispenses it into the first column of the 96 well flat bottom plate (Position B).

Without changing the GRIPTIPS, the VOYAGER begins the serial dilution by aspirating the specific volume (Figure 3) from column 1 of the 96 well flat bottom plate (Position B) and dispensing into the second column. The VOYAGER then mixes 100 μl (2- and 10- fold serial dilution) or 80 μl (5-fold serial dilutions) of the analyte/ diluent 5 times at maximum speed (10). A blowout is performed to clear the tip of any remaining liquid before aspirating for the following dilution step. The procedure is repeated until column 11 is reached, where the last aspiration is discarded along with the GRIPTIPS (Figure 4). Column 12 only contains diluent, and functions as a blank for background noise elimination.

ASSIST PLUS pipetting robot together with the 8 channel 125 µl VOYAGER adjustable tip spacing pipette is performing a serial dilution.
Figure 4: The ASSIST PLUS and VOYAGER performing serial dilution of tartrazine.

Tips:

  • Pre-wetting tips when pipetting aqueous liquids ensures excellent accuracy and precision.
  • Using adjustable mixing cycles compensates for slower mixing speeds or poorly soluble analytes.

Results

The performance of the 8 channel 125 μl VOYAGER on the ASSIST PLUS during serial dilution of 0.36 mM tartrazine in water in 96 well flat bottom plates (Figure 5) was analyzed at 428 nm absorbance using the Tecan Infinite® M200 PRO. More detailed data is provided in INTEGRA's comprehensive guide to performing serial dilution.

A 96 well flat-bottom plate with a finished 2-fold serial dilution of tartrazine.
Figure 5: 2-fold serial dilution of tartrazine in a 96 well flat bottom plate.

Figure 6 shows a representational, optimized calibration curve of a 2-fold serial dilution. Automating all liquid handling steps and mixing 100 μl of each dilution (>80 % GRIPTIPS volume) 5 times at maximum speed (10) led to reliable results in 3 independent runs. Furthermore, final values of less than 1 % were calculated for the accuracy and precision of the individual dilution steps.

Illustration of a tartrazine 2-fold serial dilution performed with optimal settings on the ASSIST PLUS pipetting robot. A linear regression plot shows the calibration curve of three independent runs.
Figure 6: Result of a 2-fold serial dilution of tartrazine using optimized mixing settings on the ASSIST PLUS.

Remarks

  • VIALAB software: VIALAB programs can be easily adapted to your specific labware and protocols, such as when partial plates are needed.
  • Partial plates: Programs can be adapted at any time to accommodate varying sample numbers, giving laboratories total flexibility to meet current and future demands.

Conclusion

  • Automated workflows on the ASSIST PLUS offer reproducible results and eliminate any operator influence on serial dilutions.
  • The ASSIST PLUS has a compact footprint to enable risk-free dilution of hazardous compounds in a biosafety cabinet
  • INTEGRA's electronic pipettes ensure homogeneity of aqueous solutions with dynamic mixing of each dilution. This is achieved by aspirating and dispensing >80 % of the total GRIPTIPS or reaction volume, repeated 5 times at speed 10.
  • Understanding how to perform serial dilutions is crucial to optimize workflows and prevent error propagation. The automated protocols on the ASSIST PLUS provide optimal liquid handling settings for 2-, 5- and 10-fold serial dilutions.

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Instruments and accessories

ASSIST PLUS, Pipetting Robot VOYAGER, 8 Channel, 125 µl Dual Reservoir Adapter Divided Reagent Reservoir, Sterile, SureFlo anti-sealing array, 25 ml, PS GRIPTIPS®, 125 µl, Sterile, Filter (for automation systems) Greiner Bio-One: 96 Well Microplate, PS, F-Bottom

ASSIST PLUS, Pipetting Robot

INTEGRA has developed the ASSIST PLUS pipetting robot to streamline routine pipetting tasks at an affordable price. Using INTEGRA electronic multichannel pipettes, the system:

  • automates pipetting tasks,
  • eliminates physical strain and
  • ensures superior reproducibility and
  • error free pipetting.

Part No. 4505

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ASSIST PLUS Pipetting Robot

VOYAGER, 8 Channel, 125 µl

VOYAGER pipettes allow the tip spacing to expand anywhere between 4.5 mm and 33 mm at the push of a button.

  • Single handed operation leaves the other hand free to handle labware.
  • On the fly access to up to 3 user-defined tip spacings.
  • These user defined tip spacings are saved. No need to memorize spacings of different labware formats.

Part No. 4722

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VOYAGER pipettes allow the tip spacing to expand anywhere between 4.5 mm and 33 mm at the push of a button.

Dual Reservoir Adapter

The dual reservoir adapter makes this system ideal for assays that required sequential addition of several low volume reagent mixes. This adapter holds two 10 ml, 25 ml or divided reagent reservoirs side by side in an aluminum frame, and can be pre-chilled to keep reagents cool during processing.

Part No. 4547

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Dual reservoir adapter with 2 didived reservoirs

Divided Reagent Reservoir, Sterile, SureFlo anti-sealing array, 25 ml, PS

Lowest Dead Volume On The Market!

25 ml divided reservoirs are comprised of 5 and 10 ml compartments, while still nesting comfortably in the standard 25 ml reusable, sturdy base. 
Each reservoir features the SureFlo™ anti-sealing array and hydrophilic surface treatment providing especially low dead volumes.

Part No. 4350, 4351, 4352

Find out more about Divided Reagent Reservoirs
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GRIPTIPS®, 125 µl, Sterile, Filter (for automation systems)

GRIPTIPS® pipette tips perfectly match the multi-lobe tip fitting, snapping firmly on during loading. This guarantees a perfect seal on every tip, preventing them from loosening, leaking or completely falling off. All tips are precisely aligned horizontally, enabling accurate touch-offs, even when pipetting with 384 tips.

Part No. 6465

Learn more about GRIPTIPS® pipette tips for Benchtop Pipetting Systems
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Greiner Bio-One: 96 Well Microplate, PS, F-Bottom

SAPPHIRE MICROPLATE, 96 WELL, PP, FOR RT PCR, WHITE, HALF-SKIRT, FOR LIGHT CYCLER, 10 PCS./BOX

Source: Website Greiner Bio One

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Customer’s voice

The ASSIST PLUS is a friendly, easy to use, liquid handling system that helps us perform the routine lab work with ease and avoiding human errors.

Sandeep Kumar, Viral Solutions