Increasing Precision When Pipetting Protein Samples: Assessing Reliability of the Reverse Pipetting Technique

Handheld pipets are commonplace in any laboratory and are used in the vast majority of applications. They are extremely diverse and are available with fixed or adjustable volumes, single or multichannels, as well as manual or electronic formats. The most commonly used handheld pipets operate via piston-driven air displacement, which moves liquid through the creation of a momentary change in pressure. The incorporation of two piston stopping points facilitates the complete dispensing of even the smallest droplets. Depression of the piston to the first of these points dispenses a volume of air equal to the volume set on the pipet. The plunger can then be depressed to the second stopping point, providing a blow-out volume, which ensures that all of the liquid volume held in the pipet is fully dispensed.

Liquid transfer process

As a seemingly straightforward process, liquid transfer actually poses a number of issues. The majority of these stem from the different properties of liquids—density, vapor pressure, viscosity, and surface tension—and how they interact with the material or the orifice bore of the pipet tip. These different liquid properties and applications therefore have a direct impact on the pipetting technique that should be used.

Liquid properties

Since the composition of any liquid influences its ability to be accurately dispensed, users need to take suitable measures to ensure that accuracy and precision are always maintained. A range of different solvents, from aqueous solutions to volatile compounds, need to be pipetted for various applications. A high liquid density will impose a greater force on the gas space between the liquid and the piston, meaning that a smaller volume will be drawn into the pipet tip. In addition, high-vapor-pressure liquids will begin to evaporate directly after aspiration into the tip. Liquids with a low surface tension, as well as viscous solutions, often leave a thin film on the walls of the tip, which will move more slowly than the rest of the liquid mass.

The selection of the correct tip, along with the right pipetting technique, can make these difficult solutions much easier to handle. Most liquid types can be dispensed using forward pipetting (Table 1). However, reverse pipetting is recommended with viscous liquids, as well as small volumes, to eliminate the effect of liquid retention on data integrity.

Pipetting technique

Pipets have been designed to ensure that accurate volumes are always effectively dispensed. Small-volume Thermo Scientific Finnpipettes (Thermo Fisher Scientific, Vantaa, Finland) incorporate a super-blow-out function to ensure the complete delivery of any microdroplets from the pipet tip. This is accomplished by the incorporation of a separate blowout piston, which delivers excess dispensing force to remove even the smallest droplets.

Figure 1 - Forward and reverse pipetting techniques.

Many biological solutions, such as bovine serum albumin (BSA), can foam up in the pipet tip or sample vessel, which can cause a deviation from the specified volume. In addition, BSA flows more slowly through the pipet tip orifice due to hydrophobic interactions with the tip material. Use of the reverse pipetting technique can improve the precision when pipetting such solutions, since it aspirates a volume that is greater than that set. The excess liquid acts as a reservoir to even out the sequential volumes, thus maintaining the integrity and subsequent reliability of data with these liquid types. This technique also prevents air from passing through the tip orifice at the end of the dispensing step, reducing the possibility of foaming. As a result, reverse pipetting is especially useful for the highly precise dispensing of small volumes. The differences between the forward and reverse techniques occur in the way in which the liquid is aspirated and dispensed (see Figure 1).

Forward pipetting:

  1. Depress the plunger to the first stopping point.
  2. Immerse the pipet tip approximately 1 cm into the liquid and slowly release the plunger to aspirate the liquid volume into the pipet tip. Withdraw the tip from the solution and touch it against the edge of the reservoir to remove any excess liquid.
  3. Gently depress the plunger to the first stopping point to dispense the liquid into the receiving vessel. Subsequently depress the plunger to the second stopping point (blow-out) to completely empty the tip of all fluid.
  4. Fully release the plunger before commencing any subsequent aspirations.

Reverse pipetting:

  1. Depress the plunger to the second stopping point (blow-out).
  2. Immerse the pipet tip approximately 1 cm into the liquid and slowly release the plunger to aspirate the desired liquid volume, plus blow-out volume, into the pipet tip. Withdraw the tip from the solution and touch it against the edge of the reservoir to remove any excess liquid.
  3. Press the plunger to the first stopping point to dispense the desired liquid volume into the vessel. Some liquid will remain in the tip (equal to the volume of the blow-out), which should not be dispensed.
  4. The remaining fluid can be returned to the original sample by depressing the plunger to the second stopping point or disposed of along with the pipet tip.
  5. Fully release the plunger before commencing any subsequent aspirations.

The precision that can be obtained when using the reverse pipetting technique to dispense BSA is discussed below.

Methods

The Thermo Scientific Finnpipette F2 pipet was used in combination with the Thermo Scientific Finntip Flex 10 pipet tips to dispense 1-μL and 10-μL volumes of 1% BSA. Each dispense was repeated five times, using both the forward and reverse techniques. The pipet was adjusted accordingly for each technique.

Results

Figure 2 - Variation in volume between forward and reverse pipetting techniques.

Figure 3 - Degree of imprecision observed with each pipetting technique for volumes of 1 and 10 μL.

As shown in Figure 2, the volume variations observed are within a much narrower range when using reverse pipetting, in comparison with the forward pipetting technique. Figure 3 demonstrates the degree of imprecision (used as a measure of the pipet’s repeatability) observed with each pipetting technique. When using the reverse pipetting technique, the results show that the imprecision was reduced by over 50% in comparison to the forward pipetting technique.

Conclusion

The properties of various liquids have a significant impact on their ability to be accurately dispensed. As such, the pipetting technique used needs to be chosen according to the liquid. Due to the initial aspiration of the selected volume plus an excess, the reverse pipetting technique significantly reduces the occurrence of inconsistencies and inaccuracies with samples prone to liquid retention within the tip. When dispensing small volumes of liquid-containing proteins, such as BSA, highly precise and repeatable resulting data can be obtained with the reverse pipetting technique.

Reference

  1. Thermo Fisher Scientific, Good Laboratory Pipetting Guide, 2010. Available at: https:// fscimage.fishersci.com/images/D16542~.pdf.

Dr. Suominen is Senior Application Scientist, and Dr. Koivisto is Research Engineer, Thermo Fisher Scientific, Ratastie 2, FI-01620, Vantaa, Finland; tel.: +358 9 329100; fax: +358 9 32910414; e-mail: info.pipettes@thermofisher.com.

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