Purpose / What It Accomplishes #
Pipetting is the indispensable technique for the accurate and precise transfer of specific liquid volumes, ranging from microliters to milliliters, in virtually all laboratory experiments. Its mastery is paramount for ensuring the reliability, reproducibility, and quantitative integrity of experimental results across all wet-lab procedures.
Principle / Theoretical Basis #
Pipettes operate primarily on the principle of air displacement or, for specialized applications, positive displacement, to accurately draw and dispense liquids. In air displacement pipettes, a piston creates a partial vacuum, which, upon release, allows ambient atmospheric pressure to force the desired volume of liquid into the tip. Positive displacement pipettes, conversely, directly displace the liquid, making them particularly suitable for handling viscous, volatile, or high-density samples where air displacement might introduce inaccuracies due to vapor pressure or surface tension effects.7 The precision of pipetting is highly dependent on meticulous technique, as factors such as temperature differentials between the pipette, tip, and liquid, the liquid’s viscosity, and surface tension can significantly influence the dispensed volume.7
Step-by-Step Explanation #
- Equipment and Reagents Required: Micropipettes (single-channel and multi-channel variants), sterile and often filtered pipette tips, the liquid samples to be transferred, and appropriate receiving vessels (e.g., microcentrifuge tubes, multi-well plates, beakers).7
- Workflow from Start to Finish:
- Volume Setting: Adjust the pipette’s volume dial to the desired setting. When decreasing the volume, it is important to approach the target setting slowly without overshooting. Conversely, when increasing the volume, it is advisable to exceed the desired value by approximately one-third of a turn and then slowly decrease to the target, again avoiding overshooting.7
- Tip Attachment: Securely attach a sterile pipette tip onto the pipette shaft. This should be done by exerting a light vertical force followed by a slight lateral rocking movement to ensure an airtight seal, critically avoiding the common practice of “hammering” the tip onto the pipette, which can damage the instrument.7
- Pre-wetting (Optional but Recommended): To enhance accuracy and precision, especially with aqueous solutions, aspirate and then expel the sample liquid back into the original reservoir at least three times. This process humidifies the air space within the tip, thereby reducing evaporation of the sample during the actual transfer and preventing significantly lower delivery volumes.7
- Aspiration: Depress the plunger smoothly and consistently to the first stop position. Immerse the pipette tip adequately into the liquid, typically about 1 centimeter below the meniscus, to prevent air aspiration. Slowly and smoothly release the plunger to aspirate the liquid. After the plunger reaches its rest position, pause for approximately one second to allow the entire liquid volume to fully enter the tip. During aspiration, the pipette should be held in a nearly vertical position.7
- Dispensing: Position the pipette tip at an angle (typically 10° to 45°) against the inside wall of the receiving vessel. Smoothly depress the plunger to the first stop, then continue to the second stop (the “purge” or “blow-out” position) to ensure complete expulsion of the liquid. After dispensing, slide the tip along the wall of the vessel to remove any residual liquid clinging to the tip’s exterior.7
- Tip Ejection: Press the tip ejector button to safely discard the used tip into a designated waste container, avoiding direct contact with the tip.7
Variations / Modifications #
Specialized positive displacement pipettes are employed for liquids that are challenging for air displacement models, such as highly viscous solutions (e.g., glycerol), volatile solvents (e.g., ethanol), or extremely dense liquids. Multi-channel pipettes are widely used for high-throughput applications, enabling the simultaneous transfer of liquids to multiple wells in microplates. Filter tips are crucial for preventing aerosol contamination, especially in sensitive applications like PCR, by creating a barrier that blocks aerosols from entering the pipette barrel.8
Applications #
Pipetting is a ubiquitous skill applied across all biotechnology laboratory disciplines. It is essential for preparing reagents, setting up diverse enzymatic reactions (e.g., PCR, restriction digestion, ligation), loading samples onto electrophoresis gels, performing serial dilutions, and managing cell cultures.5
Strengths and Limitations #
- Strengths: When performed correctly, pipetting offers high levels of accuracy and precision, which are critical for obtaining reproducible experimental outcomes. Its versatility allows for handling a wide range of liquid volumes, making it adaptable to numerous experimental designs.
- Limitations: The technique is highly dependent on the operator’s skill and consistency. Inconsistent plunger movements, improper immersion depth, or holding the pipette at an incorrect angle can significantly compromise accuracy, potentially by as much as 1% to 50% in extreme cases.7 Furthermore, improper handling, such as reusing tips or allowing liquid to enter the pipette’s interior, poses a substantial risk of cross-contamination.8
Why It Should Be Learned #
Proficiency in pipetting is a foundational competency for all laboratory work in biotechnology. Errors in this basic skill can propagate throughout an entire experimental workflow, leading to systemic inaccuracies or false results in subsequent, more complex techniques. For instance, imprecise pipetting can lead to incorrect reagent concentrations in a PCR reaction, resulting in suboptimal amplification or even failure, thereby wasting valuable reagents and time and ultimately yielding irreproducible data. The ability to execute this technique flawlessly is therefore indispensable for reliable scientific investigation.