In different fields, the term “aliquot” has different meanings: in chemistry and biology it refers to a smaller fraction of an entire sample. In the most part, an experiment only requires these fractions.
Therefore, aliquoting must be done before other productive tasks can be started. In some cases, aliquotting is just the simple transfer of the same volume from one source to numerous destinations. However, it can also involve a more complex distribution from numerous sources to numerous destinations, at different concentrations and different volumes.
The task can be further complicated if replicates are required for each different aliquot. As the complexity of the task increases, the hands-on time required from lab personnel also increases. More complex tasks also increase the risk of protocol execution errors and variability. Furthermore, repetitive aliquotting also exposes users to the risk of Repetitive Stress Injuries (RSI), as well as being costly and impacting on the quality of laboratory activities. There are many traditional automated liquid handling systems available currently.
However, systemic use of these is often neither accessible nor cost-effective for aliquotting activities. It is time-consuming to setup these expensive and complex devices and most automated liquid handlers need specific and expensive labware designed for automation. This is an unjustified extra expense if the aliquotting samples are to be used in manual experiments.
Aliquotting is probably used most commonly to extract the original stock samples into smaller portions. This means that different analytical tests of the same materials can be done, whilst at the same time preserving the original stock for future use. This is a daily occurrence at biobanks, where thousands of body fluid samples like urine, saliva and blood are delivered in tubes that need to be aliquoted into various cryo-vials.
Another common application is to aliquot master mixes of buffers and reagents during sample preparation for genetic and biochemical tests in molecular diagnostic, pre-clinical and forensic assays. These include PCR, RT-qPCR and ELISA. In these situations, aliquots are commonly taken from individual tubes and put into a range of smaller tubes, 384- or 96- well microplates.
Aliquotting can also be used to avoid repeated freeze-thaw or open-close cycle of reagents and this helps prevent changes in the physiochemical characteristics, for example heat-liable enzymes, easily oxidized chemistries of dNTPs, volatile solvents, delicate living cells and light-sensitive fluorescence conjugating moieties.
Only small aliquots of these reagents are required at a time, and so by using individual aliquots only when required, quality degradation is minimized. This in turn minimizes the negative consequences of failed experiments and the expenses that come with having to repeat work.
Most aliquotting applications only have one set volume that is dispensed repeatedly. However, some applications require volumes to be continuously changed. For example, lyophilized primer samples, which are normally synthesized at different quantities in microtubes, need to dissolved at different volumes to ensure that the concentrations of all primer stock solutions are equal. Another common example is the normalization of different protein, RNA or DNA samples to the same concentration. This requires aliquotting different volumes of buffer or water to the original sources or to different destinations.
Aliquotting variable volumes is particularly time consuming, tedious and prone to error. Human variability and simple errors in aliquotting can be very detrimental and lead to experimental results that are unusable and the waste of valuable reagents or samples.
Manual Aliquotting Completely Eliminated by Andrew
Automated aliquotting is the most convenient and obvious solution to eliminate the problems associated with manual pipetting, including pipetting mistakes, decreased lab efficiency, increased occurrence of RSI and working days that are too busy. However, most automated systems available currently are only compatible with limited consumables, specifically designed for each system, disregarding the variety of course and destination labware actually used in laboratories.
Consequently, additional transfer steps and reformatting of samples is required. Due to its highly adaptable working deck, the bench-top pipetting robot Andrew is the most suitable and affordable solution for small to medium throughput activities. The Andrew robot provides consumable flexibility that is not achieved with any other traditional robotic stations.
Andrew’s working deck is made up of an innovative system of magnetic tiles, known as Domino blocks. These can be adapted to practically any type of regular consumable used in aliquotting tasks. As a result, Andrew can aliquot in almost any arrangement defined by users and also perform complex liquid handling tasks including cross-reactions, serial dilutions, array re-formatting, normalization, cherry picking and creation of master plates.
Andrew uses the same single channel pipettes present in most life-science laboratories and is operated from the user-friendly software, Andrew Lab. It only takes a few minutes to set up aliquotting protocols using Andrew Lab and this minimizes the time spent designing protocols. Furthermore, by importing and exporting data from and to ELNs, LIMS, or any other spreadsheet format, error-free traceability and documentation can be achieved.
How to Aliquot with Andrew? Three Examples
1. Aliquotting from One Source Tube to Multiple Destination Tubes with the Same Volume
One single aspirated volume of a solution can be dispensed rapidly in multiple destination tubes, using the “Repetitive” mode of Andrew. Andrew uses standard, classical mechanical pipettes to fill up the pipette tip to the highest capacity before dispensing multiple aliquots of the same volume one after the other, without touching the liquid. Therefore, it can return to source to refill for subsequent multi-dispensing cycles, without the risk of contamination. In order to ensure both precision and accuracy, Andrew discards the first and last aliquots of the dispensed series as these are normally affected by the largest error.
For instance, three 200 mL aliquots of a set stock sample can be made in one aspiration step using a 1000 mL pipette (Figure 1). The advantage of the repetitive mode becomes obvious when this principle is applied to 18 aliquots of 50 mL done in a single aspiration step: the time needed to aliquot compared to single dispensing is significantly shorter and consumption of tips is minimized (see the Andrew Alliance Application Note on Repetitive dispensing in Reference).
Figure 1: Schematic of the Repetitive pipetting mode of Andrew.
Two common procedures exemplify the productivity of Andrew in aliquotting from one source to multiple destination tubes. These procedures, preparing aliquots of urine and whole blood samples, are commonly performed in diagnostic labs (Figure 2). Using the virtual lab bench space in Andrew Lab, the mouse cursor is used to indicate the pipetting direction from the source tube “blood” to the first destination tube “cryo”.
Figure 2: Example of an Andrew Lab protocol for aliquotting blood and urine samples with Andrew.
Holding down the Ctrl key whilst selecting additional tubes will include additional aliquotting targets. The aliquotting volume is then simply entered into the pop-up screen. The process can be repeated for aliquotting from the “urine” source tube to the four destination tubes “vial”. This takes less than two minutes in Andrew Lab. Furthermore, protocols in Andrew Lab can be saved and used again for recurrent aliquotting tasks. This improves efficiency when handling lots of destinations.
2. Aliquotting from One Source Tube to Multiple Destination Tubes with Different Volumes
Another situation that is very common in the lab is aliquotting of variable volumes to multiple destination tubes from one source. Preparing primer stocks is a good example of this scenario. Synthesis yields vary between oligonucleotide sequences and experiments and so researchers need to dissolve the lyophilized primer stocks with different volumes of buffer or water so that they are all at the same concentration. The process becomes very simple and free from errors when Andrew Lab and Andrew are used.
The process begins with the creation of the desired consumables corresponding to the source (buffer or water) and destination vials in the virtual bench space of Andrew Lab (Figure 3A). This is followed by the pipetting step which can be accomplished easily, either by clicking on the “Pipetting” action from the “Add step” panel or by dragging the mouse cursor from the source tube to one of the destination tubes. Multiple volumes can be chosen by entering the volumes of buffer to be pipetted to each primer tube in the list, which is displayed after activating the “Multi-volume mode” option (Figure 3B).
Volumes can be entered in descendent or ascendant order to optimize time by minimizing the time needed for Andrew to change volumes and tips. By copying and pasting from a list previously designed in a spreadsheet or other document, the values can be introduced all at once (Table 1). The graphical and textual description of this aliquotting activity will be shown as a single instruction (Figure 3C).
Table 1: Primer list before and after being sorted according to volume.
Figure 3: Main steps in designing an aliquotting experiment from one source to many destination tubes in Andrew Lab.
Andrew can also be used to automate the resuspension step that follows. This is done by introducing a mixing action at the destination tubes, under the “Mixing” panel. The speed of mixing can be adapted (fast, medium or slow), to fit the experimental requirements or user preference. Introducing this mixing step can remove the need for the vortexing and centrifuging steps that are often required during resuspension of the primers. This substitution leads to better reproducibility and saves time. This aliquot protocol only took two minutes to design, and seven minutes to execute using Andrew (with the “on-the-fly” mode of pipetting activated).
For a total aliquot number of 100, the hands-on time increases to 10 minutes for protocol design and an additional five minutes to set up the consumables for Andrew. Manual aliquotting of 100 different volumes however, takes about 50 minutes of hands-on time to complete. Therefore, primer stick preparation using Andrew reduces hands-on time for repetitive aliquotting (using a pre-designed experimental protocol) by a factor of 10 (from 50 minutes to five minutes, Figure 4).
Figure 4: Workflow for the preparation of primer stocks either manually or by using the pipetting robot Andrew.
This is still the case, even if tubes need to be manually uncapped and accommodated using the Andrew physical work bench. Automation also allows more samples to processed in a reproducible and uniform manor.
3. Aliquot from many Source Tubes to many Destinations with Replicates
Technical replicates as well as biological repeats of the sample are critical in real-time PCR experiments. These replicates are essential for the establishment of the experimental error due to the amplification and analysis technique, and this allows confidence levels to be set for what is meant to be significant data. However, the preparation for the experiment becomes more cumbersome and complex because it involves aliquotting activities that are time consuming and need to be done extremely carefully.
Therefore, automating this task is desirable. Eight different DNA samples from four different biological conditions with two biological replicates each are tested in this example. For each sample, three technical repeats are included, resulting in the preparation of 24 aliquots in total, from variable sources (Figure 5). Moreover, these reactions need to be randomly distributed in a 96-well microplate in order to reduce bias error.
Figure 5: Experimental design of a real-time PCR experiment including three technical replicates per sample.
Similar to the example above, the volumes to be pipetted are sorted in order to reduce the volume changes, and consequently the execution time of the experiment. Next, the source and destination tubes are dropped in triplicates from the virtual bench to the consumable section (Figure 6A). Then, the two lists, of the destination well positions and volumes, are copied and pasted into the corresponding Andrew Lab fields (Figure 6B).
All of the 24 aliquot pipetting steps are included in one instruction (Figure 6C). Normally, Andrew will change the tip for each pipetting step. However, if the user wishes to reduce consumable costs, fewer tips can be used (for example one single tip for each set of triplicates). In this instance, the protocol can be designed with an independent pipetting instruction for each sample, and this gives the user full control over tip choice for the aliquotting of each sample (Figure 7).
Figure 6: Design of an aliquotting experiment involving many sources and many destination wells in Andrew Lab. A) Source and destination tubes are dropped in the Andrew Lab virtual bench. B) The list of volumes to be pipetted and destination wells positions can be directly introduced or simply pasted from a spreadsheet into the Andrew Lab columns dedicated for this information (green highlighted area). C) At the end of this procedure, all pipetting steps are consolidated in a single instruction (yellow highlighted area).
Figure 7: Aliquotting triplicates in an independent pipetting step for each source tube with Andrew Lab.
If the sources also come from multiple wells in another microplate, or even in the same microplate, the entire source plate can be dropped into Source. The sorted list of source well positions, corresponding to the correct aliquot volume, can then be copied and pasted during the Configuration step. If a consumable representing a rack of tubes in any format is dragged onto the virtual bench, the same procedure can be followed. This makes designing the experiment easier and faster.
This experiment takes about 5.5 minutes to design in Andrew Lab, if the tips are changed for every single aliquot. Andrew completes this protocol fully unattended and with no mistakes in only 10 minutes. Although executing the protocol manually may take approximately the same amount of time, manual execution is prone to possible errors and is entirely dependent on the operator.
The robot Andrew improves aliquotting efficiency significantly by fully eliminating human variations and errors, minimizing labor time, adapting to a diverse range of labware and integrating multiple aliquotting strategies. The intuitive Andrew Lab software facilitates simple-to-design protocols and improved documentation and traceability of the aliquotting steps and materials.
In addition, the personal health of the user is protected by avoiding the direct handling of chemically or biologically hazardous liquids and the risk of developing muscular disorders such as RSI is reduced (see the Andrew Alliance Application Note on pipetting ergonomics in Reference).
- Q.A. Ngo. Automatic accurate repetitive pipetting with standard pipettes by Andrew robot. Application Note No. 6 – 20160418 v3. Andrew Alliance. 2016.
- J. Denyes & N. Pennese. Pipetting Ergonomics: risks and solutions. Application note No. 1 - 20160912 v2. Andrew Alliance. 2016.
About Andrew Alliance S.A.
Andrew Alliance is an independent, privately financed company, based in Geneva, Boston and Paris. The company was created in March 2011.
Andrew Alliance is dedicated to advance science by working with scientists to create a new class of easy-to-use robots and connected devices that take repeatability, performance, and efficiency of laboratory experiments to the level required by 21st-century biology.
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