Proper pipetting: 10 tips on how to pipette
Proper pipetting: 10 tips on how to use a pipette
Are you annoyed with inaccurate pipetting results? Learn how to correctly use a pipette and improve your results instantly. This article explains the following ten tips in detail:
- Ensure temperature equilibrium: Make sure the pipette, tips and liquids are at room temperature (if experiment allows)
- Maintain consistent pipette angle: Hold the pipette at a consistent angle not exceeding 20 degrees
- How to aspirate: Aspirate by immersing the tips just below the liquid’s surface (2-3 mm)
- Touch off after dispense: Touch off after each dispense when removing the pipette from the target vessel
- Pre-wet: Pre-wet the pipette tip by aspirating and dispensing the nominal volume 3 times
- Optimize the volume range: Pipette within the optimal volume range of your pipette (35-100 % of the nominal volume for air displacement pipettes)
- Discard first and last dispense: When dispensing multiple aliquots, it is recommended to discard the first and last dispense of the series
- Viscous liquids: Pipette viscous liquids at slower speeds and in "Reverse pipet" mode
- Volatile liquids: Pipette volatile liquids quickly and in "Reverse pipet" mode
- Calibrate based on liquid density: Recalibrate your pipette if the liquid has a considerably different density than water
Before you start
1. Ensure temperature equilibrium
To improve accuracy and precision when using an air displacement pipette, the pipette, tips and liquids need to be equilibrated to room temperature if the experiment allows.
Accuracy refers to how close the dispense volume is to what you set the pipette to dispense. It is affected by pipette calibration and pipetting technique.
Precision measures how consistent the volume is over numerous dispenses. Precision can be corrected with proper pipetting practices.
Note: Temperature differences lead to volume contraction or expansion of the air cushion inside the pipette tip and pipette, which can negatively impact the accuracy and precision of the dispense.
How to pipette
2. Maintain consistent pipette angle
When pipetting, the angle of the pipette can play an important role in obtaining good results. If possible, hold the pipette at a constant angle throughout the entire process. Ideally, the angle at which the pipette is held should not exceed 20 degrees. For very small volumes of 30 microliters or less, the straighter the pipette, the better.
Note: With changing the angle, the hydrostatic pressure inside the tip varies. As a result, the aspiration volume will be inconsistent.
3. How to aspirate
A common mistake made during aspiration is to immerse the pipette tip as deep as possible in the source vessel. As this increases the risk of liquid droplets clinging to the outside of the pipette tip, it is best to immerse the pipette tip just below the liquid’s surface. It is recommended to insert the tip only 2-3 mm into the source liquid to allow the desired volume to be aspirated. An exception to this is the aspiration of extremely low volumes such as 0.2 microliters.
Note: Liquid retained on the outside of the tip can result in an inaccurate dispense.
4. Touch off after dispense
After a dispense, you will often see a droplet at the end of your tip. As this droplet belongs into the dispense, you should use one of the following three methods to remove your pipette from the target vessel.
- Side wall touch off
Remove the pipette tip by sliding the tip end along the sidewall of the vessel. This is the standard method and recommended to achieve the most accurate dispense.
- Surface touch off
Remove the tip by touching off the liquid droplet on the surface of the liquid in the container. This technique is recommended when dispensing less than 1 microliter as a neat transfer. Touching the droplet to the liquid draws the small droplet out of the pipette tip which ensures accurate delivery.
- Into liquid dispense
If the dispense was made directly into the liquid it is considered a wet-dispense and a touch off is not required. This method is ideal for small volume dispensing to ensure the liquid does not remain on the side of the vessel.
Note: After a dispense, residual liquid often clings to the tip end. The above techniques remove this residual liquid from the tip.
Optimizing pipetting performance
After loading tips onto your pipette, aspirate and dispense the nominal volume 3 times. This will equilibrate temperature differences and humidify the dead air space inside the pipette and tip. When neglecting the pre-wetting procedure, the first few dispenses tend to deliver less volume due to evaporation.
Note: The evaporation can also cause droplet formation on the tip end, as vapor pressure increases and liquid is forced out of the tip.
6. Optimize the volume range
The volume range of your pipette can have an impact on both accuracy and precision. Air displacement pipettes show the best performance between 35 % and 100 % of their nominal volume. The closer your dispense volume is to the total volume of the pipette, the better your results. Below 35 %, the volume of dead air in the pipette becomes quite large, and the risk of inaccurate and imprecise dispensing increases. On top of that, pipetting within the optimal volume range is less technique dependent and reduces user related errors.
7. Discard first and last dispense
When using the repeat dispense mode of an electronic pipette, it is recommended to discard the first and last aliquot of the series. Here's why:
- When the electronic pipette is finished aspirating the liquid, the mechanics inside the pipette need to change direction before starting to dispense. This could result in the first dispense being too low in volume.
- As no pipette is 100 % accurate, each dispense might be slightly off the target volume. The last dispense will include the accumulated error of all previous dispenses.
Note: The first and the last dispenses should not be used for the assay because they contain the largest errors.
8. Viscous liquids
Viscous samples can be challenging to pipette. They usually enter the tip more slowly than other liquids and tend to stick to the tip wall when being dispensed because of their low elasticity. Therefore, you should apply the following techniques when pipetting viscous liquids:
- Viscous samples should be aspirated and dispensed at slower speeds. Moverover, pausing after every aspiration or dispense gives the liquid more time to smoothly move into or out of the tip.
- To avoid that you dispense volumes that are too low because of liquid adhering to the tip's inside wall, you need to use the "Reverse pipet" mode. As it aspirates the selected volume plus an extra dispense that will be discarded, it is ideal to compensate for the retained liquid. An additional benefit of reverse pipetting is that there is no blow-out at the end. This prevents that viscous liquids start to foam or form air bubbles.
9. Volatile liquids
Volatile liquids can be difficult to pipette accurately. As they evaporate faster than aqueous solutions, tips often start to drip. To prevent dripping tips, you should pre-wet your pipette tip to humidify the dead air space. This can be achieved by aspirating and dispensing the full volume of your tip three times using the liquid you will be pipetting. Additionally, volatile solutions should be pipetted quickly and in “Reverse pipet” mode. The "Reverse pipet" mode incorporates a larger sample volume to minimize the effect of evaporation on the actual volume to be delivered.
10. Calibrate based on liquid density
Significant errors can occur when liquids other than aqueous solutions are used. Recalibrate your pipette if the liquid has a considerably different density than water.
Note: Pipettes are normally tested and calibrated at the factory with distilled water at room temperature. Pipetting liquids with different densities results in inaccurate dispenses.