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

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

Step-by-step procedure

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.

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.

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.

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.

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.