About Liquid Handling

Knowing the type of liquid that is being handled is essential to achieving proper and accurate liquid transfer. While a lab technician can correct for manipulation errors and readily adapt his/her movements and practices during manual pipetting, automated liquid handling systems (Andrew+ and Pipette+) are totally agnostic to the properties of the liquid and the way it should be handled.
Therefore, all commands must be well-configured upfront to fitting liquid handling requirements and ensure efficient and consistent pipetting performance during execution. This involves implementing adequate pipetting parameters via the laboratory software OneLab and feeding the information into the system to guarantee full control of every pipetting step throughout the experiment.

This reference guide provides insights into liquid classes and the appropriate techniques to use for handling each liquid class, ensuring precise and consistent pipetting. Additionally, It offers numerous recommendations to optimally set up the OneLab protocol for successful liquid transfer using the Andrew+ Pipetting Robot or the Pipette+ Guided System.

Liquid Classification

  • 💦 Aqueous Solutions: water, TRIS, DMSO, buffers with a pH of 7 such as PBS, liquids containing small amounts of detergent, acid/base solutions, diluted nucleic acid, or protein samples.

  • 🍯 Viscous Solutions: glycerol, oil, bead slurry, non-ionic surfactants such as polysorbate or Tween 20.

  • 💨 Volatile Solutions: methanol, ethanol, isopropanol, acetone, formaldehyde, acetonitrile.

  • 🩸 Body Fluids: whole blood, serum, plasma.

  • 🧬 Biological Samples: genomic DNA, RNA, PCR products, cells.

  • 🧼 Foaming Liquids: protein-rich solutions (e.g. BSA-solution or cell culture medium), anionic surfactants such as sodium dodecyl sulfate or SDS.

Pipetting Recommendations

The two tables below summarize pipetting recommendations for the six liquid classes above-mentioned. The parameters represented in the table are key for the optimal configuration of OneLab to correctly handle each liquid type. To define the best liquid handling settings for a given solution used in your method, always choose the liquid class with attributes that are the closest possible to the solution of interest. For instance, if you are preparing a Master mix, follow the recommendations regarding Aqueous Solutions as the Master mixture is part aqueous and will have similar properties.

💦 Aqueous Solutions

🍯 Viscous Solutions

💨 Volatile Solutions

Pipetting mode

Forward

Reverse (1);

Forward with air cushion control is an alternative

Forward with air cushion control; Reverse is an alternative

Pipetting speed

Normal to fast

Slow to avoid the formation of air bubbles

Fast to minimize the effects of evaporation

Tip pre-wetting (2)

Optional

Not recommended

Highly recommended to reduce dripping

Air cushion

Not required

Top air cushion (3) with forward pipetting

Bottom air cushion (4) with forward pipetting

Dispense tip position (5)

On the fly, at the liquid level, or at the bottom

Ideally at the liquid level or at the bottom especially if the consumable already contains other liquid

On the fly, at the liquid level, or at the bottom

Pause before blow-out (6)

Not required

Helpful especially in forward pipetting mode when low-retention tips are not used

Recommended

Pause after aspiration/dispensing (7)

Variable

Recommended to ensure full deliver

Not required

Touch off after dispensing (8)

Optional

Helpful to remove residual liquid

Not required

Rinse after dispensing

Not required

Not possible

Not possible

Repetitive dispensing (9)

Suitable

Not suitable

Not suitable

Tip type

Standard

Standard, ideally low retention

Standard

Tip filter

Not required

Not required

Recommended to eliminate exposure to harmful vapours and protect pipette integrity

🩸 Body Fluids

🧬 Biological Samples

🧼 Foaming Liquids

Pipetting mode

Forward;
Reverse mode is an alternative

Forward

Reverse;

Forward mode is an alternative

Pipetting speed

Slow to avoid splashing

Slow to normal; Avoid rough and excessive pipetting (11)

Slow to avoid foaming and bubble formation

Tip pre-wetting (2)

Not recommended

Not required

Not recommended

Air cushion control

Not required

Not required

Not required

Dispense tip position (5)

At the liquid level or at the bottom

At the liquid level or at the bottom

Ideally, at the liquid level or at the bottom especially if the consumable already contains other liquid; On the fly is feasible in some cases

Pause before a blowout (6)

Not required

Not required

Helpful especially in forward pipetting mode when low-retention tips are not used

Pause after aspiration/dispensing (7)

Recommended to ensure full deliver

Not required

Recommended to ensure full deliver

Touch off after dispensing (8)

Helpful to remove residual liquid

Helpful to remove the residual liquid; Liquid surface touch off is recommended for dispensing small volumes

Helpful to remove residual liquid

Rinse after dispensing

Dispense into the liquid with rising (10) is ideal to clear the interior wall of the tip

Rinse the tip after dispensing to make sure no material is sticking to the interior surface of the tip

Rinse the tip after dispensing for a complete transfer when the forward pipetting mode is used

Repetitive dispensing (9)

Can be used with diluted samples

Suitable; Helpful for aliquoting

Suitable

Tip type

Standard

Standard, but sterile for cell cultures, ideally low retention for nucleic acids

Standard, ideally low retention to avoid loss of sample or reagent

Tip filter

Recommended to eliminate contamination of the lower part of the pipette

Recommended to reduce aerosol formation and cross-contamination

Not required

(1). Reverse pipetting

The liquid is over-aspirated and then only the precise volume is dispensed into the destination consumable without blowout (purging the tip). In the case of viscous solutions, the excess volume aspirated helps compensate for the retained liquid that adheres to the tip's inside wall, while in the case of volatile solutions, the use of a larger volume aims to further reduce the effect of evaporation on the actual volume to be delivered. The remaining/extra volume in the pipette tip is either:

  • Discarded with the tip to avoid contaminating stock solutions or study samples (this option is not available yet in OneLab),

  • Dispensed in a specific waste container before discarding the tip which from a safety standpoint is essential when handling biological, microbial, radioactive, or toxic substances (this option is not available yet in OneLab),

  • Pipetted back into the source container in the case of precious reagent aliquots only if no tip touch occurred during transfer.

One drawback of reverse pipetting is that minimal dripping can still occur during transit with some types of liquids mainly volatile solutions. In this case, reverse pipetting decreases the error in the delivered volume but does not eliminate dripping.

(2). Pre-wetting

Involves aspirating and immediately dispensing liquid several times (at least 3 times) prior to aspirating the desired volume for liquid transfer. It is an easy technique to improve the accuracy of liquid transfer in different ways:

  • Saturates the air inside the pipette tip with vapours from the volatile solution leading to a stabilization of the pressure above the liquid column and consequently reduction of liquid dripping during the transit,

  • Neutralizes the capillary effect in micro-volume pipetting,

  • Pre-conditions the inner surface of the pipette tip and equalizes the temperature of the air inside the tip with that of the sample or solution to be transferred.

(3). Top air cushion

Is a volume of air aspirated before the liquid and then dispensed last to completely expel the liquid in the tip, ensuring total delivery of the desired volume. This is very helpful for dispensing viscous solutions and low volumes. Be careful when dispensing is performed at or below the liquid level, the release of the air cushion at the end of the dispense may cause bubbles or aerosols to form. Please note that this option is only available with the forward pipetting mode.

(4). Bottom air cushion

Is a volume of air aspirated after the intended volume to dispense providing a greater air buffer to hold the liquid in the tip, preventing droplet formation and dripping during transit. Please note that this option is only available with the forward pipetting mode.

(5). Dispense tip position

This parameter determines the position of the pipette tip with regards to or in the consumable when dispensing the liquid into the destination consumable:

  • On the fly: Indicates a non-contact dispense, which is preferable to dispensing directly at the liquid level or to the bottom of the consumable in order to avoid cross-contamination between transfers or liquid carryover due to accumulation of the solution between the tip end and the consumable surface. The pipette tip is never in touch with the sample or solution in the destination consumable and therefore very convenient for dispensing buffer solutions or media for cell cultures. In some cases, the one-the-fly mode can increase the accuracy of the reserve dispensing by reducing the chance that excess volume gets drained off the tip. Keep in mind that the on-the-fly mode is not recommended for small-volume dispensing of 10 µL or less.

  • At liquid level: Dispensing is made at the surface or a few mm below the liquid's surface (2-3 mm).

  • To the bottom of the consumable: This mode is particularly convenient to dispense small volumes. Use bottom touch-off during dispensing to reach the very bottom of the consumable and subsequently ensure delivery of all the intended volume and that no liquid droplet is adhering to the end of the pipette tip. Dispensing to the bottom of the consumable is not recommended when the bottom surface area is coated with target molecules such as immobilized antibodies or affinity proteins, or occupied with growing cells as attached monolayers in culture.

(6). Pause before a blow-out

This action is recommended when pipetting volatile solutions. It allows time for any remaining liquid to collect and settle at the bottom of the pipette tip prior to purging, thereby expelling all the liquid into the destination consumable. This ensures accurate delivery of the intended volume. It can also be helpful for viscous solutions that tend to adhere to the inner surface of the tip to help evacuate as much liquid as possible from the pipette tip when using forward pipetting with a top air cushion.

(7). Pause after aspiration/dispensing

It is critical for the aspirated liquid to reach equilibrium with the air in the tip prior to dispensing. Therefore, a seconds-scale pause time is helpful and consists in maintaining the tip in the liquid before removing it completely. The time duration depends on the volume and the liquid type being aspirated. This is also applicable to dispensing. Liquids that are highly viscous or deposited at the bottom of an empty consumable require a longer pause after dispensing to ensure full delivery of the volume. Most liquids do not require a long pause after pipetting, especially volatile solutions that require a very short pause to minimize the amount of time the tip is in contact with the liquid and prevent droplet formation.

(8). Touch off after dispensing

After a dispense, a small liquid droplet often clings to the end of the tip. The surface tension between the liquid and the tip material often prevents all of the liquid from being fully expelled during dispensing. Two touch-off techniques can be used to assist in draining the residual liquid out of the pipette tip, ensuring accurate delivery of the desired volume:

  • Consumable sidewall touch-off (this option is not available in OneLab)

  • Liquid surface touch off

The standard method of "sidewall touch-off" consists of removing the pipette tip from the solution by sliding the tip end along the sidewall of the consumable allowing the residual liquid to be more easily removed from the tip end. This method is not feasible with Andrew+.

On the other hand, the method "liquid surface touch off" involves removing the tip from the solution by touching the remaining droplet to the surface of the liquid in the destination consumable, which draws the small droplet out of the pipette tip. The liquid touch-off method is recommended when dispensing small volumes (less than 1 µL). In OneLab, this manoeuvre can be achieved through a custom tip position which consists in adjusting the dispensing height with respect to the liquid surface in the destination consumable.

(9). Repetitive dispensing

Uses the maximum capacity of the pipette to aspirate enough liquid from the source and subsequently dispense the same volume (if only the repetitive mode is selected and a single volume is defined) or different volumes (if the repetitive mode is combined with a multivolume dispensing) over multiple destinations at one time without changing the pipette tip in between. The first and the last aliquots are discarded as they contain the largest errors.

While repetitive dispensing can save time and tips, enabling rapid coverage of the destination consumable, it is less precise than forward dispensing. It requires a good knowledge of liquid properties to ensure a precise liquid transfer. When using the repetitive mode to sequentially dispense into multiple empty target wells or tubes, the dispense tip positions "to the bottom of the consumable" or "at the liquid level" are generally recommended compared to the "on-the-fly" option for improved pipetting accuracy and consistency. Please keep in mind that repetitive dispensing is not suitable neither for viscous nor volatile solutions.

(10). Dispense into the liquid with rinsing

This technique is ideal for heterogeneous samples, such as blood and serum to ensure that the full sample is delivered for accurate downstream processing. The dispense is made directly into the liquid in the destination vessel sufficiently below the liquid surface. After slow dispensing, the tip remains in the solution and several pipetting up/down cycles are performed until the interior wall of the tip is clear (rinsing). Once done, the tip is slowly removed from the solution by sliding it along the interior wall of the destination consumable to avoid liquid carryover. Finally, the tip is purged by a blowout to completely empty the tip.

(11). Avoid rough and excessive pipetting when handling biological materials, particularly genomic DNA and cells

One should avoid rough and excessive pipetting to prevent undesirable damage due to shear force, thereby preserving the viability of cultured cells and the integrity of high molecular weight DNA during sample preparation. Wall shear occurs within a pipette tip at the plane of contact between the sample fluid and the inside wall of the tip.


Check out the "OneLab Pipetting Tips & Tricks" articles to:

Set up OneLab parameters for accurate pipetting of viscous solutions
Set up OneLab parameters for accurate pipetting of volatile solutions

Set up OneLab parameters for accurate pipetting of foaming solutions

Learn more about the OneLab protocol designer and pipetting parameters

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