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The skill of using a micropipette is crucial for molecular biology researchers. Without good skills, your experiments will be difficult to replicate, stock solutions will be inaccurate, and analyses will have significant errors and become meaningless. However, fortunately, you don't need to worry too much about this. The following article will help you improve in this area, and once you grasp these principles, you will enhance the accuracy of your pipetting as desired.

General Structure of a Pipette

Most of us use conventional pipettes (Air Displacement Pipette)

A conventional pipette operates somewhat like a syringe, except that there is an air gap between the piston and the sample. This air gap prevents the piston from touching the solution. While keeping the solution and pipette separated in this way is beneficial, it also imposes some limitations on the pipette.

Temperature and pressure can affect the volume of the air column, impacting the accuracy of the pipette. Volatile solvents can also evaporate into the air column, leading to inaccurate or reduced volumes when dispensed. The pipette's air column can become contaminated when aspirating solutions. If you need to work with corrosive substances or hazardous biological materials, this is a real issue.

Other Types of Pipettes (Depending on Your Application)

Most of the information in this article pertains to conventional pipettes; however, in certain circumstances, a piston pipette (positive displacement pipette) is a more optimal choice. A piston pipette operates like a syringe but without the air column, making them more accurate for aspirating volatile solvents. The absence of an air column also reduces contamination risks when aspirating corrosive or hazardous biological materials, making these pipettes more suitable for such solutions.

These types of pipettes are also quite expensive since the pipette body and the tip are integrated and discarded together after use. To save costs, you might use conventional pipettes with filter tips, although this introduces some other issues.

PIPETTE MAINTENANCE

Pipettes need to be serviced every 6-12 months

Pipette maintenance includes recalibration, lubrication of parts, and replacement of worn or other parts. You can send pipettes to companies for maintenance.

Regularly inspect pipettes for damage

Clean pipettes with 70% alcohol before use

Use a pipette holder when not in use

Always hang pipettes vertically on a stand to allow water/condensation to drain out, preventing corrosive solutions from flowing back into the pipette and causing damage.

Never invert a pipette with a filled tip

Nothing can prevent the solution from flowing back into the pipette body, causing contamination or damage.

SELECT THE RIGHT TIP TO INCREASE ACCURACY AND REPEATABILITY

Tips and pipettes that do not fit well together can reduce pipetting accuracy. It is recommended to use pipettes and tips from the same system. Regularly check the pipette tip connection to ensure a proper fit.

GOOD PIPETTING PRACTICES

Ensure you know how to use the pipette correctly

You can refer to the pipette manuals corresponding to the type of pipette you use. Remember the most important rules:

Consistency when aspirating:

Use the pipette smoothly.
Apply consistent pressure on the plunger.
Be steady and gentle.
Hold the pipette vertically at a 90-degree angle to the liquid surface when aspirating.
Submerge the pipette tip gently and adequately when aspirating solutions.

ASPIRATION ANGLE

Wait time for small volume pipettes:

Keep the tip submerged in the solution and wait at least 1 second after aspirating the sample.
Then slowly withdraw the tip from the solution.
This is very important for large volume samples and high-viscosity samples.

DISPENSE TECHNIQUES

There are three techniques for dispensing:

Dispense against the side of the container (1).
Dispense on top of the existing solution surface (2).
Dispense into the solution (3).

Note:

Pause before dispensing with viscous samples.
If dispensing into the solution, keep the plunger at the second stop position until the tip is withdrawn from the solution. Avoid drawing the sample back into the pipette.

CHECKING PIPETTE ACCURACY

Check pipette accuracy by aspirating 100 µl of water and weighing it; the weight should be around 0.1g. Repeat this 10 times and record the weights. If the variation is more than ±0.5%, either recalibrate the pipette or practice your pipetting technique more.

TIPS TO IMPROVE PIPETTE ACCURACY

Pre-rinse the pipette tip

Pre-rinsing the tip ensures that all internal parts are in contact with the solution. Do this by aspirating the sample into the tip and then dispensing it back or into a waste container to rinse the tip, which helps increase the accuracy of your pipetting process. It is recommended to use this method for volumes greater than 10 µl.

REVERSE PIPETTING TECHNIQUE

This technique is used for aspirating viscous solutions (e.g., blood) or volatile solvents. It is also useful for aspirating very small volumes such as 0.5 µl or less.

Reverse pipetting begins by pressing the plunger to the second stop, thus including an extra volume in the aspiration process that is not included during the final dispense.

CONSIDER THE AMBIENT TEMPERATURE

Individuals or companies usually calibrate pipettes at room temperature, so if you use a pipette at a different temperature (e.g., in a cold room), you may not achieve accurate volumes.

CONSIDER SAMPLE TEMPERATURE

In a recent article in Nature Methods, Millet and Barthlen observed a strange phenomenon: when repeatedly aspirating cold samples, the dispensed volume in the first dispensation is always larger than the aspirated volume, but subsequent aspirations with the same tip yield accurate volumes. This is also true for hot samples, except that the first dispensed volume is smaller than the aspirated volume. Therefore, they proposed a simple solution: dispense the first aspiration back into the sample container and start from the second aspiration.

USE A PIPETTE SUITABLE FOR THE VOLUME

The accuracy of a pipette decreases when the dispensed volume approaches the minimum volume the pipette can handle. For example, if you want to aspirate and dispense 15 µl, using a 1 ml pipette is a terrible choice, and a 200 µl pipette is not much better. Ideally, use a 20 µl pipette.

VOLUME RANGE SPECIFICATIONS: 10-100% OF THE PIPETTE'S VOLUME

RECOMMENDED VOLUME RANGE

Generally, 35-100% of the specified volume.
Below 35% depends on high technique. Using volumes below 10% of the pipette can affect accuracy by more than 3%.

USE THE LARGEST POSSIBLE VOLUME

Using a pipette with a larger volume always yields more accurate results than using a smaller volume. Accurately aspirating a small amount is not easy and can introduce statistical errors into your results. To solve this problem, you can dilute the stock solution about 10 times and use a larger volume pipette to aspirate the solution.

SETTING THE PIPETTE

Adjust the pipette more than 1/3 turn beyond the desired volume. Then adjust back to the desired volume. Perform this operation for different volumes and at different times. This can reduce the effect by 0.5%.

COMMON PIPETTING ERRORS

Source: BioMedia Vietnam