Nucleic acid quantitation is an important step in molecular biology workflows, particularly for advanced applications such as qPCR and NextGeneration Sequencing (NGS). In NGS, the ultimate goal is to generate highly complex libraries sequenced to the greatest degree of depth and uniformity, making accurate quantitation is especially important. Two critical steps for accurately determining nucleic acid concentration are: 1) Before library preparation where maximum enzymatic efficiency is sensitive to DNA input amount and 2) After library preparation to normalize samples for pooling prior to sequencing. In addition, the desire for everlower sample input (e.g., for RNA-seq experiments) and the challenge of isolating nucleic acid from many common sample types such as FFPE and ccfDNA make estimating concentration difficult.
While a variety of quantitation methods exist, fluorescent dye-based systems are recommended for advanced downstream applications because of their sensitivity, target selectivity, cost, and ease of use. With the QuantiFluor® dsDNA System, the fluorescent dye stock is diluted to form a working solution, added to a plate that contains a small volume of purified DNA sample and fluorescence is measured using a fluorometer. The sensitive and accurate QuantiFluor® dsDNA System can be used for quantitating dsDNA in single tubes as well as 96- and 384-well plates.
Accuracy during repetitive pipetting of small volumes can be difficult and labor intensive with many opportunities for error and costly re-work. Therefore, most labs scaling to higher throughput choose to adapt their quantitation assay for an automated robotic liquid handling platform. The MANTIS® Liquid Handler is a novel bench-top liquid handler that can meet many requirements for high-throughput reagent dispensing at a fraction of the cost of larger systems. For nucleic acid quantitation, the MANTIS accurately dispenses small volumes of the QuantiFluor® Dye working solution for reproducible quantitation results. In this application note, we demonstrate the compatibility of the MANTIS liquid handler to quickly and accurately automate reagent handling of the QuantiFluor® dsDNA System for both 96- and 384-well plate formats.
Figure 1. Quantitation of K562 human genomic DNA standard (Cat.# E4931) using the MANTIS® Liquid Handler to dispense the QuantiFluor® dsDNA dye working solution in 96-well plate format (Panel A) and 384-well plate format (Panel B). Fluorescence was measured on a GloMax® Discover System and unknowns were interpolated from a power regression standard curve.
Figure 2. Quantitation of a 2,200bp PCR product used to mimic the average size of a mRNA transcript. The MANTIS® Liquid Handler was used to dispense the QuantiFluor® dsDNA System in a 96-well plate format (Panel A) and 384-well plate format (Panel B). Fluorescence was measured on a GloMax® Discover System and unknowns were interpolated from a power regression standard curve.
The MANTIS liquid handler provides researchers with a flexible workstation for setting up various protocols in minutes. These protocols are executed precisely to enhance laboratory efficiency and experimental reproducibility. This application note demonstrates how easy liquid handling software is to use, providing quick and seamless transition to an automated platform. Adding the reagents for the QuantiFluor® dsDNA System using the MANTIS liquid handler offers reproducible quantification in multiwall plates with virtually no instrument programming.