Perform serial dilutions on OT-2

Abstract Serial dilutions are an important component of various biological experiments. For example, generating a standard curve for DNA, protein, or cell culture is a use case that requires diluting the sample with a constant dilution factor. Here, we present an alternative to performing these repetitive steps of manual pipetting. The opentronOT-2 robot can serially dilute 8 samples in approximately 2 minutes, with 10 dilutions having high reliability between replicates. The difference in accuracy expressed by the coefficients per column is comparable to that of more expensive liquid handling platforms. Users are encouraged to adapt the attached parameterized protocol to their specific scientific applications.

Introduction Serial dilutions are both stand-alone experiments and building blocks of more complex experimental pipelines. Applications can be as simple as generating a standard curve or more complex phenotypic characterization across cell culture concentrations. In any application, precise pipetting is required to produce reliable results. In general, manual laboratory work can introduce random errors into any pipetting step in the protocol. These pipetting inaccuracies can include incorrect dilution factors. Pipette mixing speed and tip height can improve accuracy, but are challenging to maintain during manual pipetting. Additionally, repeated pipetting can aggravate the damage over time. OT-2 is an alternative option to ensure pipetting reliability and ultimately data quality for serial dilution experiments.

Materials - 2 Liquid Handling Robot - 2 Software Version 3.1.2 - Phototube P300 Multi-Channel Pipette - Vision Center 300µLTip Rack - American Scientific 12 - Channel I Automated Reservoir (1061-8150) - Corning 96 Well EIA/RIA Easy Clean TM Clear Flat Bottom Polystyrene (3368) - Corning 500 mL Molecular Biology Grade Water (46-000-CV) - Assorted Food Color and Egg Dye by McCormick & Co. - Blue

• Tecan unlimited F200 pro

Protocol 1. Prepare a blue dye solution using 50 µL of blue food dye and 10 mL of molecular biology grade water. 2. Add 200 µL of the blue dye solution to the A1-H1 weIIs of the 96-weIIlat bottom nail agent. 3. Add 20 mL of diluent to hole A1 of the trough reservoir. NOTE: The column after the last dilution is the default position for the blank area. The last well of the tank/reservoir is the default liquid waste. 4. Arrange laboratory cutlery in slots 1-3 according to the deck layout in Figure 1a. - Slot 1 = Vision Center 300µLtip Rack - Slot 2 = 12 Channel Reservoir

• SIot 3 = 96-weIIlat bottom pin
• Please change the parameters in the protocol to the following:
• Dilution factor = 1.5
• Final volume = 200
• Number of dilutions = 10
• Continue to the OT-2 Run application, upload your protocol and calibrate the labware.
• Seal the plate and quantify the absorbance at 450 nm, 25 lashes per pin reader.

Results Figure 1 The average raw absorbance value of the frst6 column is shown in Figure 2. Each column shows 1.5 times dilution than the previous column. Consistency of variance (CV%) illustrates high reliability between replicates. Table 1 shows columnar CV% for each dilution plate. The average precision per column was about 2%, which is comparable to the value of similar experiments using a liquid handler that is much more expensive than the OT-2. Each serial dilution plate was completed on the OT-2 in about 2 minutes.

Conclusion Here, we present a simple serial dilution experiment illustrating the high-precision pipetting of the OT-2 liquid handling robot. In addition to precision, the OT-2 is capable of highly customizable pipetting techniques. Users can optimize pumping/dispensing speeds, mixing speeds, air gaps and many other options for more accurate and efficient liquid handling. Automating serial dilutions will provide scientists with a reliable pipetting system and valuable walking time away from tedious, error-prone manual work.