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

Here we show how to do serial dilution of tartrazine in water with an 8 channel 125 µl VOYAGER adjustable tip spacing pipette on the ASSIST PLUS pipetting robot.

Experimental set-up

The ASSIST PLUS pipetting robot, together with the 8 channel 125 µl VOYAGER adjustable tip spacing pipette and 125 µl sterile, filter GRIPTIPS®, automates complete serial dilutions in one program consisting of three 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 dilution with the ASSIST PLUS pipetting robot

Step-by-step procedure

Serial dilution of an analyte

The INTEGRA dual reservoir adapter together with two 10 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 adjustable tip spacing pipette (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 two (Position B). For each dispense, a pre- and post-dispense with 5 % (10-fold serial dilutions) or 10 % (2- and 5-fold serial dilutions) of the transferred volume is used to guarantee precision during plate set-up.

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 one of the 96 well flat‑bottom plate (Position B) and dispensing into the second column. The VOYAGER mixes then 100 µl (2- and 10-fold serial dilutions) or 80 µl (5-fold serial dilutions) of the analyte/diluent five times at maximum speed (10). Before aspirating for the following dilution step, a blow out is performed to clear the tip of any remaining liquid. 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 blank for background noise elimination.

Tips:

• Excellent accuracy and precision with tip pre-wetting when pipetting aqueous liquids.
• Adjustable mixing cycles to compensate slower mixing speed or poorly soluble analytes.

Results

The performance of the 8 channel 125 µl VOYAGER adjustable tip spacing pipette on the ASSIST PLUS pipetting robot during serial dilution of 0.36 mM tartrazine in water in 96 well flat‑bottom plates (Figure 5) was analyzed at 428 nm absorbance (Tecan Infinite® M200PRO). Detailed data is provided in our comprehensive guide on how to perform serial dilution.

Figure 6 shows a representational, optimized calibration curve of a 2-fold serial dilution. Automating all liquid handling steps and mixing each dilution with 100 µl (>80 % GRIPTIPS volume) five times at maximum speed (10), led to reliable results in three independent runs. Furthermore, final values of below 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 pipette, labware and protocols, for instance when partial plates are needed.
• Partial plates: Programs can be adapted at any time to a different number of samples, giving laboratories total flexibility to meet current and future demands.