As modern researchers increasingly work with high-value, small-volume samples, the need for advanced pipetting techniques/protocols is evident. In order to accurately detect microscopic changes in small sample volumes, test tubes and vials are increasingly being replaced by microtiter plates, which require reagents in the microliter/nanoliter range.
When working with small volumes, factors such as temperature and dispensing accuracy are critical to control and operation. If you want consistent results when working with small amounts of liquid, you need to identify and eliminate all potential sources of error and regularly review your laboratory workflow. This applies to errors caused by using the wrong pipetting instruments and inaccurate pipetting techniques. Here are some tips for accurately pipetting small volumes.
Pipetting tips
Using the right pipette and pipette tips
Since volumetric errors are more prominent at low sample volumes, it is important to choose pipettes that provide a sample volume consistent with the study protocol. Make sure you choose the smallest possible pipette for the sample volume. For example, if you are pipetting a 1 µL sample, use a 2-5 µL pipette rather than a 20 µL pipette.
In addition, you need to know if you are using the right type of pipette for your workflow. For example, volumetric pipettes are ideal for volatile, viscous, and other types of difficult samples. It is recommended that you read the manufacturer's instruction manual when choosing the right type of pipette for your experimental needs. Most errors reported in the laboratory are due to volatility or viscosity of liquids and unexpected environmental changes. You must ensure that your pipetting technique is consistent with the surrounding conditions, sample type, and sample volume.
Using the Right Pipetting Technique
There are various considerations when working with smaller volumes of liquid using a particular pipetting technique. For example, you need to ensure that you keep the pipette in the proper vertical position and pull the pipette straight out of the sample container while avoiding contact with the sides of the container.
For sample volumes less than 50 μL, avoid holding the pipette at an angle as it can alter the amount of liquid aspirated. In addition, if you allow the pipette tip to touch the side of the container, it will create a wick process that will reduce the amount of liquid dispensed.
In addition, it is important that you always pause after pipetting and before removing the tip from the liquid. In addition, try to use consistent plunger pressure and speed. Depress the plunger smoothly until it stops, dip the pipette tip, and then release it at a constant rate. This is recommended as such repetitive operations may produce reproducible results.
Consider various ambient temperatures
You need to make sure that you design your pipetting workflow with various environmental factors in mind, including the temperature, pressure and humidity levels in your laboratory. Experts recommend that you should not pipette small volumes at temperatures below the ambient laboratory temperature. The ambient temperature of the laboratory will directly affect the precise volume of samples pipetted by the instrument, as thermal expansion caused by higher temperatures may increase the air space between the sample and the pipette tip and significantly reduce the dispensing volume.
Equilibrating small volume samples in a laboratory setting can help significantly reduce volume errors and prevent over-dispensing of cold liquids. In addition, you can use positive displacement pipettes when working with very cold or very hot liquids to maintain a higher level of precision and accuracy.
Pre-wetted pipette tips
In order to properly aspirate and dispense small volumes of sample, you must pre-wet the pipette tips before starting the pipetting workflow. Pre-wetting primarily involves immersing the pipette tip in liquid before performing subsequent pipetting steps.
Failure to pre-wet the pipette tip can result in increased evaporation and reduced delivery despite the use of proven pipetting techniques. Pre-wetting the pipette tip allows you to dispense an accurate and precise amount of liquid by increasing the humidity level in the chamber, thereby limiting liquid evaporation during pipetting.
Use the correct immersion depth
It is important to properly immerse the tip below the meniscus before aspiration. For large-volume pipettes (1-5 mL), the immersion depth should be at least 5-6 mm, while for smaller-volume pipettes, the depth should be around 2-3 mm.
If you submerge too little, especially with large-volume pipettes, air may be drawn in. On the other hand, if you submerge too much, it can cause the liquid to stick to the surface of the pipette tip, leading to volumetric errors.
If you ignore any of these variables, the chances of errors sneaking into your workflow can increase dramatically, leading to unreliable results. As sample volumes get smaller and smaller, the effects of their physical properties, ambient temperature and pipetting technique become more prominent, making pipetting workflows quite daunting. Follow better practices and pay attention to a few key aspects
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