Suction and release the micropipette properly

What is a micropipette?

A micropipette is an indispensable tool in modern science, especially in the fields of molecular biology, cellular biology, medicine, and chemical analysis. They allow for precise transport of small liquid volumes, typically measured in microliters, ranging from 0.1 µL to 1000 µL. With micropipettes, experiments can be conducted with high accuracy and reproducibility.

Pipetting cycle

To be concise and more familiar with the terminology in Vietnam, GeneSmart will use the term “pipette” to refer to the term “micropipette.” In this article, GeneSmart only discusses the most commonly used air displacement pipette.

Pipetting cycle step-by-step:

1-Select an appropriate volume range pipette.

2-Adjust the selector wheel to the desired volume.

3-Tip loading.

4-Liquid aspiration (depress, hold and release plunger).

5-Liquid dispensing (depress, hold and release plunger).

6-Tip ejection.

Pipetting technique

The pipette is designed with 2 stops, the first stop (neutral stop) allows for liquids to be aspirated and/or dispensed, while the second stop (blowout stop) controls the blowout onto a designated vessel. There are 2 different yet powerful techniques when using air displacement pipettes: forward and reverse pipetting. Forward technique can deliver volumes accurately when pipetting aqueous solutions, while reverse pipetting is highly recommended when dealing with challenging liquids (e.g., viscous, dense).

Forward technique uses the first stop to aspirate and dispense liquid (Figure XX). The Forward technique is not accurate when pipetting volatile or high viscosity liquid. The high evaporation pressure of volatile solvents causes immediate dripping from the tip. Both vapor and droplet will make the inside volume decrease. Meanwhile, the default force of the first stop is insufficient to aspirate the required volume if the liquid has high viscosity. The Reverse technique aims to overcome these issues.

Reverse technique uses the second stop to aspirate and the first stop to dispense liquid (Figure XY). When using the second stop, the volume aspirated will be higher than the desired volume. However, dispensing at the first stop ensures that the dispensed volume remains accurate. The extra volume left in the tip will be discarded along with the tip.

Common tips to get better pipetting results

1/ Immersion Angle: Keeping the immersion angle close to vertical can improve accuracy by up to 2.5% in microvolume pipettes. Keep the pipette within 20° of vertical – anything greater can affect measurements.

2/ Immersion Depth: Immersing tips to the correct depth improves accuracy by up to 5%, so use the recommended depths shown here. Immersing too deep can cause too much liquid to be aspirated. Conversely, positioning the tip too close to the surface can aspirate air. Both result in inaccurate volume.

3/ Pre-rinsing: Pre-rinsing can increase accuracy by up to 0.2%. It helps neutralize capillary effects in microvolume pipettes and, for large-volume tips, equalizes the air temperature inside the tip with the temperature of the sample.

4/ Dispensing: Improve accuracy by up to 1% with good dispensing technique. For highest consistency, touch the vessel wall with the tip to release any remaining sample, then slide it up the wall to prevent liquid from clinging to the orifice. The two other techniques are “dispensing into liquid” and “dispensing onto liquid surface”, and they also work well with aqueous (non viscous) liquids.

5/ Rhythm and Speed: Consistent pipetting rhythm and speed will improve accuracy by up to 5%. Fast or “jerky” aspiration can cause splashing, aerosols, shaft contamination and loss of sample volume.

6/ Setting the Micrometer: When changing the volume to a lower setting, dial down to the desired volume setting. But when changing the volume from a lower to a higher setting, turn the selector wheel about 1/3 turn above the desired volume setting, then slowly dial back downto the desired setting. This will avoid “mechanical backlash” and assure greater accuracy.

7/ Volume vs. Range: Improve accuracy and precision by up to 1% by aspirating and dispensing samples within 35% to 100% of a tip’s nominal volume. Avoid setting a pipette’s volume to less than 10% of its maximum.

8/ Hand Warming: When pipetting over long periods, heat from your hand can cause air inside the pipette to expand and produce inaccurate results. Avoid the effects of hand-warming by placing the pipette on its stand between pipetting cycles, instead of holding it in your hand.

9/ Temperature: An ideal temperature for pipetting is 21.5°C (±1°C). Your instrument and samples should be the same temperature as the room in which you’re working, so allow them to equilibrate for 20 minutes if necessary. Also avoid drafty or sunlit areas that can cause large or sudden temperature changes. Pipetting at a constant temperature improves results by as much as 5%.

10/ Ergonomics: Practicing good ergonomics improves accuracy and performance. Hand and body fatigue leads to errors, especially when dispensing large numbers of samples.

• Good posture reduces fatigue and the potential for injury
• Pipettes with finger-hooks allow you to relax your grip regularly
• Switching hands periodically reduces fatigue and increases productivity
• Rainin XLS pipettes offer extremely light spring forces and our patented LTS™ LiteTouch™ tip ejection system – essential safety equipment!