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Pipetting accuracy is how close the volume actuall
Hardly any activity is as common in laboratories as pipetting. Whether PCR, ELISA or dilution series – liquids need to be moved at virtually any time in most labs. Pipetting is therefore often considered a supposedly simple routine activity and receives little attention. Yet to use the right tools and follow proper technique is crucial for reproducible analytical results.
Tip1: Start with choosing the right pipetting system for the job
Pipetting errors do not only occur during dosing, but already beforehand. Many researchers use common air-cushion pipettes for any pipetting task – although these can only inadequately handle challenging liquids such as e.g. Glycerol in high concentrations. Another example: Filling a series of 40 tubes with an air-cushion pipette – a very strenuous task.
In both cases, a positive displacement system with dispensing function (like the Multipette® from Eppendorf) would be better suited to decrease pipetting errors or improve your pipetting efficiency. This shows that you should always start with thinking about the right pipetting system for the task at hand. Keep in mind this rule of thumb: Air-cushion pipettes are ideal for liquids that behave like water, while positive displacement systems are more suitable for liquids with varying viscosity, volatility, surface tension or density as well as hot or cold liquids. Think about electronic pipettes if you’re dealing with several samples and looking for improved ergonomics, a higher precision as well as an additional efficiency gain due to various operating modes (such as e.g. pipetting and dispensing with only one tool). For more guidance regarding suitable pipetting systems for different tasks see this chart from Eppendorf.
Tip 2: Follow proper pipetting technique when using air-cushion pipettes
Air-cushion pipettes are the most common dispensing system in labs around the world. Follow these six steps to employ them correctly and increase the reproducibility of your results:
Step 1: Make sure to always work close to the nominal volume of your pipette – that’s where accuracy and precision are highest because the air cushion inside the pipette is as small as possible. Step 2: Hold the pipette as vertically as possible and don’t change the angle when aspirating liquid. This especially applies to multi-channel pipettes, which many users hold at an angle while aspirating liquid from reservoirs. Step 3: Immerse the pipette tip as little as possible into the liquid. The tip should only be immersed a few millimeters – just enough to prevent air uptake.
Step 4: Pre-wet the pipette tip by taking up and discharging the liquid several times before transferring the volume to increase accuracy. Prewet the tip at least three times to equilibrate the air to the liquid. Depending on the liquid type and volume, more pre-wetting steps might be helpful.
Step 5: It takes some time for the liquid to completely flow into the tip: Wait a few seconds until you pull the tip out of the liquid. We recommend counting from 20 to 23 for small volumes and to 25 for large volumes before pulling the tip out.
Step 6: For liquid dispensing, there are fewer individual factors to consider. However, it is very important, that the control button of a mechanical pipette is really held down until the tip has left the liquid. A lot of routine often leads to a merging of the individual steps “pulling out” and “sliding the control knob back” into one movement. It is then easy to inadvertently draw some liquid from the target vessel back into the tip
Tip 3: Don’t forget that pipetting systems consist of a pipette and a suitable pipette tip
Many researchers underestimate the influence of pipette tips on the dispensing results.
The mere fact that a tip fits onto a pipette cone is no guarantee for the performance of the total system encompassing the pipette and the tip. Even the best pipettes cannot produce reliable results if pipette tips with flaws in production quality or material are used. Therefore we recommend using high-quality pipette tips from the same manufacturer as your pipette. If pipette tips from a different manufacturer are to be used, you should always calibrate your pipette accordingly. Learn more in Application Note 354 from Eppendorf.
Tip 4: Pay attention to the lab environment and adjust your (air-cushion) pipette if needed
When using air-cushion pipettes, a difference in temperatures between the instrument, tip, liquid and room can adversely affect your pipetting result. The same applies if your lab is situated at high altitudes above sea level. Therefore, you should make sure that the pipette, tips and liquids are at room temperature (if your experiment allows for it).
When pipetting at high altitudes you should adjust your pipettes following the standard operating procedures as defined by the manufacturer. Some electronic pipettes (such as the Eppendorf Xplorer®) come with a pre-programmed adjustment setting for different heights.
Tip 5: Calibrate your pipetting system in regular intervals Have your dispensing system calibrated regularly! Pipettes contain wearing parts such as sealing rings on the tip cones that are worn out over time. The effect is not noticeable for a long time – unless you maintain and calibrate the pipettes regularly. The international standard for pipettes ISO 8655 recommends to re-calibrate pipettes at least yearly, depending on your workflow and lab procedures it might be necessary more often. Professional service providers (such as epServices from Eppendorf) provide validated (ISO 17025) and reliable services according to ISO 8655 to help you maintain premium performance.
If you want to learn more and dive deeper into the science of pipetting to perfection, we recommend downloading this comprehensive eBook from Eppendorf for free
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