Liquid Handling FAQ

The pipet tip tends to drip when liquids like ethanol, acetone or chloroform are used. The cause of the dripping is the high vapor pressure of the liquid that leads to an expansion of the air cushion inside manual pipettes. One way to avoid dripping is pre-wetting the pipet tip. You have to aspirate and dispense the liquid 2-3 times prior to transferring the liquid into your destination vessel. Another option is to use a different pipetting system, the so called positive displacement system. It is without an air cushion and therefore no dripping occurs.

No, cutting the pipet tip is the worst thing to do when transferring glycerol. The pipetting result will neither be precise, nor accurate. The pipet tip is deformed and the orifice will be frayed so that plastic residues might also fall into the sample. It is better to use a full pipet tip and reverse pipetting technique or a positive displacement system.

Eppendorf solutions:
Eppendorf ViscoTip^®

Some liquids contain a large amount of protein, like BSA-solution or cell culture medium. These tend to build up foam during pipetting, especially in the moment of blow-out when air is introduced into the sample. To avoid this phenomenon one should apply reverse pipetting technique with air cushion pipettes. Another option is the usage of positive displacement systems. These lack the air cushion and therefore no foam can build up during dispensing of such liquids.

Pipetting detergents such as Triton X-100 or Tween 20 lower the surface tension of aqueous solutions. Furthermore they tend to stick to the inside of a pipet tip after dispensing. That means that never all the liquid inside the pipet tip is dispensed, leading to sample loss, inaccurate volume delivery and in the long run higher reagent costs. Efficient pipetting of detergents is only possible with pipet tips specifically developed for these liquids or a positive displacement system.

Eppendorf solutions

• Eppendorf ViscoTip^®
• Eppendorf Combitips advanced^®
• epT.I.P.S.^® LoRetention

Aerosols can be formed by almost every liquid. If the air humidity inside the pipette is below liquid humidity small microdrops evaporate upwards into the pipet tip and up into the pipette cone. If the liquids used are warm e.g., cell culture medium, the evaporation increases. The inside of the pipette cone can suffer from corrosion over time. But even worse is the risk of contamination brought into samples via aerosols. If an infectious liquid or bacterial culture evaporates into the pipette cone you will dispense the infected and contaminated aerosols with the blow-out. It is possible that you cross-contaminate multiple samples. Preventing aerosols is necessary and can be achieved by using high quality two-layered filter tips that block fine aerosols, bacteria and viruses. If you are using highly infectious liquids a positive displacement system might be more advantageous because the whole sample is enclosed in the tip without any contact to the liquid handling instrument.

When using a two-button pipette you have to aspirate and dispense the liquid with one button. Then you switch over to a second button for tip ejection. Liquid aerosols ascend up into the pipet tip and even higher into the pipette cone during aspiration. Then the liquid is dispensed and you aspirate air again when releasing the operation button. Thereby you may aspirate the aerosols again, prior to tip ejection via the second button. With a one-button operation pipette you aspirate and dispense the liquid and then eject the tip in the same downwards movement. So you do not aspirate aerosols back into your pipette.

Liquids with a high density such as sulfuric acid or phosphoric acid have a tendency to pull down the air-cushion inside the pipette. Thereby the air-cushion is elongated which leads to lower volume aspiration than set on the pipette. The pipetting result is always inaccurate without having the pipette adjusted to the higher density. After adjustment the pipette aspirates a little bit more liquid due to a smaller air-cushion. The pipetting result is correct then.

An air-cushion pipette is the most known pipette type. It is used together with pipet tips. Inside the pipette and the pipet tip is air of the surrounding area, the so called air-cushion. This air is moved downwards when pressing the operation button and the sample liquid is aspirated due to the partial vaccuum inside the pipette. The sample liquid is then dispensed by the air pressure when the operation button is pressed down again. The blow-out at the end of a pipetting event is a little amount of extra air that is blown out to deliver all liquid out of the pipet tip.

A positive displacement system is mainly used for dispensers, but also some pipettes exist that work according to this principle. Since the instruments themselves do not have a piston, a special tip with an integrated piston is needed to complete the system. When the tip is inserted into the instrument the piston is coupled and gets pulled upwards during usage. The sample liquid is aspirated directly without any air between the tip's piston and the liquid. With this system some difficult liquids like glycerol, ethanol, blood or cell culture medium can be easily aspirated and dispensed.

A positive displacement system can be used for any type of sample. But it is recommended especially for viscous, volatile, dense, detergent- and protein-containing liquids. Since it lacks the air-cushion, more precise results can be achieved than with classic pipettes. Often positive displacement systems are so called dispensers. So when working with plates it is advantageous to use a dispenser. With this instrument the whole plate can be filled with only one aspiration, but multiple dispensing steps.

Reverse pipetting means uptake of liquid including the blow-out volume and dispensing of liquid without blow-out volume. Reverse pipetting is therefore recommended to reduce the error, resulting in more volume being aspirated than necessary. The excess aspirated solution remains in the tip and is discarded.

Regardless of the volume, the air-cushion should be wetted by multiple filling and emptying of the tip before dispensing. This saturates the air cushion and leads to more accurate and reproducible results.

For viscous liquids we recommend positive-displacement instruments like Biomaster, Multipette and Varipette or an air-cushion pipette (Research plus, Reference 2) in combination with reverse pipetting technique (see userguide 20 and 21 for details).

Eppendorf solutions

• Eppendorf ViscoTip^®
• Eppendorf Combitips advanced^®

Polyethylene glycol is very viscous. Therefore, instruments with positive displacement principle can be used. These systems have an integrated piston in the tip and do not have an air-cushion. Thereby, they are capable of dealing with problematic liquids.

Eppendorf solutions

• Eppendorf ViscoTip^®
• Eppendorf Combitips advanced^®

For chloroform we recommend positive-displacement instruments or an air-cushion pipette in combination with pre-wetting and reverse pipetting technique (see userguide 20 and 21 for details or download the poster "Pipetting techniques" ).

Diethyl ether has such a high vapor pressure that it would leak out of the tip immediately using an air-cushion pipette. For this reason, it can only be pipetted using a positive-displacement pipette.

Due to the increased vapour pressure of methanol we recommend to pre-wet at least 3 times and perform reverse pipetting technique.

Both dichloromethane and n-hexanol have a very high vapor pressure. Using a normal air-cushion pipette would lead to dripping of the liquid and inaccurate volume delivery. For that reason positive displacement instruments should be used.

Following contamination of pipettes with radioactive liquids, we recommend putting the contaminated parts in a complex-forming liquid or special cleaning solution. After that, rinse very thoroughly with distilled water and let dry. Grease piston lightly afterwards.

An adjustment of air-cushion pipettes is required for solutions with a density > 1.2 mg/µL. Note that variable manual pipettes turn into "fixed-volume pipettes" when being adjusted. They can only be adjusted for one volume and not across the entire volume range. Sophisticated manual pipettes have a user adjustment for temporary adjustment purposes. This user adjustment can easily be set back to manufacturer settings without the need for recalibration.

The RFID chip is used to clearly identify an instrument. Identification data, e.g. serial number or results of quality control test of manufacturer, are stored on the RFID chip. These data can not be changed or deleted.

Tips with low retention properties will grant an advantage especially during pipetting of solutions containing detergents, such as PCR master mixes, enzyme solutions and many buffers. These tips are characterized by significantly improved flow performance, thus achieving more economic use of expensive reagents, while valuable sample material is used effectively. Furthermore, reproducibility is improved, since wetting of the tip is dependent on both user and handling (for example: pipetting speed) and can therefore vary considerably.

The self-sealing mechanism works best with aqueous solutions, most acids and bases. Strong acids and bases might not be blocked properly and increase the risk of damaging the pipette if accidental over-pipetting occurs. For very aggressive solutions we recommend positive displacement systems where the sample is secured in the tip without any contact to the liquid handling instrument.