Rapid Quantification and Normalization of DNA for UV Measurement

Simplifying and speeding up DNA Quantification and Normalization using the Fluent Laboratory Automation Solution from Tecan.

Introduction

The quantification and normalization of DNA are essential steps after nucleic acid purification, in the majority of genomics processes. These need to be incorporated into all workflows in this field, therefore.

Absorbance techniques have been the standard for all types of applications in this field, owing to its wide flexibility and ability to find the concentration of all types of nucleic acid with the help of their specific empiric extinction coefficients.

This is where the Fluent Laboratory Automation Solution and FluentControl™ software are proving their worth in adding power and ease to workflows in genomics procedures. This article sets forth the benefits of this system in terms of its Smart Commands feature, which ensures simplified methodology, absence of manual calculations and better ease of adaptation to specific workflows. This includes the following command options:

  • Store Well Concentration: this menu command records each well sample concentration after accounting for any potential dilution
  • Normalization: this uses recorded data to generate a description of the required concentration and volume for each sample in the plate being used or in a new plate
  • Reagent distribution and Sample transfer: this makes the process of preparing the quantification plate and normalized sample plate quick and easy

The Fluent platform for liquid handling has an inbuilt microplate reader. This allows the system to be set up, following which no operator input is required to generate the quantification and normalization results.

In addition to this simplicity, the process is completed in 25 minutes or less, including nine minutes to set up the quantification plate, five minutes to take the UV measurements, and nine minutes to aspirate samples from the normalized sample plate with pipettes.

The Pathlength Correction feature helps enhance the accuracy of calculation of the DNA concentration with either a prespecified correction factor or the reader’s automatic correction function, for pathlength.

The intuitive TouchTools™ touchscreen is a user interface that actually tells the operator what options can be selected (volume or concentration) as well as instructions on setting up the workstation. This makes sure that the procedure is compliant with SOPs as well as makes the process secure against unwanted sources of result variability.

Materials and methods

This experiment uses an automated set up for UV quantification testing, namely, the Fluent platform which has an Air FCA™ that uses filtered single-use tips, an InfiniteR M200 microplate reader and a Robotic Gripper Arm™, all operated by the FluentControl software with the above SmartCommands.

As the first step prediluted (1:10) samples were added to a 96-well UV-StarR half-area plate (from Greiner Bio-One) and made up to a volume of 100 µl before measurements were taken.

A sample containing Herring sperm genomic DNA from Promega (D1815) with its concentration unknown was measured a total of eight times over five separate runs, to evaluate the reproducibility of the workflow between runs and within a single run. The samples were laid out in checkerboard fashion at high and low concentrations for this testing.

A triplicate assay was performed to generate a dilution curve at final concentrations with a range of 0.98 to 125 µg per ml in the tested microplate, in order to determine the linearity of DNA measurement.

Workflow

The user-friendly UI of the FluentControl software helps the operator complete the set up as shown in Figure 2, and define without difficulty the options to be selected, namely:

  • Sample number between 1 and 96
  • Sample dilution ratio for quantification (in this experiment it was set at 1:10)
  • Final normalized sample volume
  • Final normalized volume concentration

The total time for this workflow even with reference measurements being taken at 260, 280, 310, 900 and 970 nm was under 25 minutes.

The Normalization Smart Command is also capable of being set to initiate a prespecified action when samples are beyond the concentration limits, or to ask for a user-specified action when such a sample is tested.

Analysis and results

Each replicated sample had its concentration calculated for all five runs with due accounting for the automatic correction for pathlength, as Table 1 shows. This result remained significantly invariable both within and between the five runs with the overall CV being less than 2%. One such DNA standard curve generated on the Fluent system is shown in Figure 4.

The results of quantification of a 96-well plate pipetted in a high concentration-low concentration checkerboard layout, with 48 samples each at high and low concentrations. The consistency of these samples was high across the plate with the CV of each sample type being 1.76 and 1.81% respectively, ruling out the occurrence of cross-contamination as seen in Figure 5.

The samples having been quantified were now normalized to have a final concentration of 250 µg per ml. They were then diluted to 1:10 and repeat quantification performed, to test the Normalization Smart Command function’s reliability. Overall, the normalized samples showed a measured concentration of 25.4 µg/ml on average, the CV being 2.56%.

Summary

The article presents proof of the high reliability and reproducibility of the Fluent automated laboratory solution for UV quantification and normalization of DNA. Its user-friendly TouchTools interface makes it simple to set up operations and also offers great scope for quick adaptation to specific workflows, as are required in post-purification quality control processes on nucleic acid. If an MCA with 96 or 384 channels is employed for pipetting, the total run time is decreased still further.

Acknowledgements

Produced from materials originally authored by Dr. Beatrice Marg-Haufe, product manager, Tecan.

References

  1. Tecan Application Note: Optical pathlength correction for absorbance measurement (Doc. No 400192_V1.0.pdf).

About TecanTecan

Tecan is a leading global provider of automated laboratory instruments and solutions. Their systems and components help people working in clinical diagnostics, basic and translational research and drug discovery bring their science to life.

In particular, they develop, produce, market and support automated workflow solutions that empower laboratories to achieve more. Their Cavro branded instrument components are chosen by leading instrumentation suppliers across multiple disciplines.

They work side by side with a range of clients, including diagnostic laboratories, pharmaceutical and biotechnology companies and university research centers. Their expertise extends to developing and manufacturing OEM instruments and components, marketed by their partner companies. Whatever the project – large or small, simple or complex – helping their clients to achieve their goals comes first.

They hold a leading position in all the sectors they work in and have changed the way things are done in research and development labs around the world. In diagnostics, for instance, they have raised the bar when it comes to the reproducibility and throughput of testing.

In under four decades Tecan has grown from a Swiss family business to a brand that is well established on the global stage of life sciences. From pioneering days on a farm to the leading role our business assumes today – empowering research, diagnostics and many applied markets around the world


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Last updated: Apr 1, 2019 at 5:06 AM

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