Concentration quantification and normalization are critical at the start of many genomic, proteomic and genetic applications, for example qPCR, q-RT-PCR, NGS library preparation, QC of protein and antibody production, sequencing, genotyping, forensics, clinical diagnostics etc.
During normalization, manual pipetting requires changing of pipet volumes for both diluent and sample, especially in medium-throughput applications using microplates. This can be very tiresome. Mistakes can easily be made during the calculation of the required transferred volumes and through dispensing the wrong liquid amount into the wrong destination. Moreover, there is unavoidable performance variation within and between human operators. This creates additional, non-biological errors, in downstream steps which results in the costly rework of the entire process.
These mistakes and the problems they cause can be avoided completely using the Normalization wizard in the Andrew Lab software in combination with the robot Andrew (Figure 1). The only thing that is required is the user spreadsheet, containing the sample location with the original volumes and concentrations, either in a set of tubes or multi-well plates. The complete pipetting protocol can be generated by a few simple clicks in Andrew Lab.
This automatically calculates the sample and buffer volumes required to achieve an equal concentration in all samples. Once the samples and buffer are provided on the working deck of Andrew, the simple “Start” button initializes the robot. It will then execute the normalized protocol, with no mistakes, from any source to any destination of common labware such as multi-well plates, tubes, vials or microtubes.
The normalized samples are then ready for use immediately in downstream applications. There is absolute certainty that they are of the correct concentration. All individual pipetting steps and intermediate liquid concentrations can be traced easily at any step. They can also be exported into documentation that conforms to the Good Laboratory Practice guidelines.
Figure 1: Simplified normalization procedure by Andrew Lab and the robot Andrew
Andrew Decreases Hands-On Time by 16 Times and Improves Reproducibility by 1.5 Times with Respect to an Experienced Human Operator
Using a photometric assessment method with the Ponceau S dye, the normalization performance of five Andrew robots (four 1000G and one 1000R model) with three different Gilson pipette sets on different days, were evaluated. Each Andrew was programmed to (a) generate 96 dye solutions at 96 different concentrations (18.5 – 95 mg/L) by mixing a stock dye solution (100 mg/L) with distilled water in a 96-well plate to achieve 25 µL, and (b) normalize the resulting 96 dye concentration into another 96-well plate to the same concentration of 18 mg/L for a volume of 150 µL. The volume range that Andrew transferred using the four pipettes P20, P100, P200 and P1000 and Gilson’s Diamond tips was 12.5 – 237.5 µL.
One Andrew 1000G was tested for reproducibility by creating 96 different concentrations for four replicated plates. The CV values for each of these 96 concentrations from these four plates show uniformity in the majority of the samples (within 1%). This indicates excellent pipetting reproducibility (Figure 2).
Figure 2: Andrew reproducibly created 96 different concentrations
Next, five different units were tested for the reproducibility of normalizing 96 different concentrations in a source plate into another destination plate at a specific final concentration. The CV of the 96 normalized concentrations performed by Andrew was computed. The automated pipetting by Andrew was compared with data generated by a human operator with 18 years of manual pipetting experience who repeated the same normalization procedure using the same pipette set.
Figure 3: Andrew required significantly less hands-on time while improving reproducibility of normalization
All five Andrews performed with superior and more uniform CVs (1.15% - 1.4%). These results were significantly better than those performed by the human operator (1.95%) (Figure 3).
When using Andrew Lab and the robot Andrew, the total hands-on time for the user was five minutes. This consisted of 2.5 minutes of protocol designing and 2.5 minutes arranging the samples and buffer onto the Andrew bench space. This is compared to the manual normalization procedure which took approximately 1 hour 20 minutes of hands-on time.
Despite the fact that each Andrew took approximately 1 hour 30 minutes to complete the normalization protocol, there was a 16x improvement in the hands-off time for the user because the Andrew works completely unattended (Figure 3).
Simplified Designing of Normalization Protocols in Andrew Lab
Users can copy and paste the list of sample concentrations, sorted from lowest to highest or vice versa, from the spreadsheet into Andrew Lab. Sorting the data expedites the execution of Andrew because less time is needed to change pipettes and volumes. Positions in the source consumable and samples volumes also need to be included in the list. The Normalization wizard of Andrew Lab generates any normalization protocol within two minutes. All it takes is six simple steps which the user can execute in a few clicks.
Step 1: Copy and paste the source names, their locations, and the initial volumes and concentrations.
Step 2: Indicate the consumables that contain the samples in order to inform the wizard of the input format (Figure 4).
Step 3: Select the method of normalization as either: (A) at a fixed volume in different destinations (Figure 5A), or (B) at variable volumes in the same source (Figure 5B). For both cases, the desired normalized concentration must be indicated. It is important to note that the wizard is aware of the practical limits of what is physically possible, depending on the source and destination capacity.
Step 4: If method 3(A) is chosen, select the destination consumable type and positions (Figure 6); if method 3(B) is chosen, the wizard skips to Step 4 because the source consumable and destination consumable are the same.
Step 5: Indicate the Buffer for sample dilution (Figure 7).
Step 6: Validate and accept the newly created normalization protocol (Figure 8).
The pipetting steps and transferred volumes of the samples and buffer can be calculated by Andrew Lab once the normalization protocol is generated. These can then be verified as standard pipetting actions within Andrew Lab. For instance, it is possible to:
- Verify protocol steps (Figure 9):
- Verify the sample volumes transferred (Figure 10):
Additionally, users can modify other protocol parameters, for example tip positions, tip choice, and mixing at the source and destinations. Users can find more details on using the Normalization wizard of Andrew Lab in the Andrew Lab manual, or by watching two real-time videos that describe the two methods:
http:/andrewalliance.com/application_notes/nor- malization1, http:/andrewalliance.com/application_notes/normalization2.
The pipetting robot Andrew is a tireless assistant for scientists and technicians. The Andrew robot will transform concentration normalization from a tedious procedure to a painless process, and boasts numerous benefits:
- Automatic pipetting decreases the risks of repetitive strain injury of human workers significantly.
- Reproducible and accurate pipetting in complex patters with random and systematic errors lower than that of a human operator, therefore improving quality of work flows.
- Decreasing hands-on time for users by 16 times for each normalization process, therefore allowing multi-tasking and enhanced lab productivity.
- Wide compatibility with diverse lab consumables in large volume range of 0.2 ml – 10 mL, using existing samples and providing ready-to-go formats for immediate downstream applications.
- Automatic calculation of the complex list of dilution volumes directly in Andrew Lab and delivery of a ready-to-execute normalization protocol, without the possibility of protocol design errors.
Even the most complex normalization protocols only take five minutes of hands-on time to design. This means that users can spend time completing other value-added activities, with the assurance that all samples will be normalized reproducibly, accurately and with a performance superior to that of most expert human operators.
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.
Start with meeting customer needs, end with customer feedback.
Andrew Alliance delivers solutions that are focused on customer needs, both today and in the future. Our products are manufactured to the highest standards, using a range of carefully selected, proven, and sustainable technologies, that ensure both high performance and reliability. We actively seek continuous customer feedback, in order to guarantee the best possible design outcomes.
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