Although each specific base has a maximal absorbance at a slightly different wavelength, on average, nucleic acids as a macromolecule will absorb maximally very near 260 nm. The aromatic ring structure of the purine and pyrimidine moieties that make up the nucleoside bases of DNA and RNA are responsible for absorbance of UV light at 260 nm (Figure 1). Differing side groups for all five different nucleosides are indicated by R1 and R2. Aromatic purine and pyrimidine ring structure of nucleoside bases. The direct quantitation of nucleic acids using the spectrophotometric absorbance at 260 nm in conjunction with the appropriate extinction coefficients (9) remains the easiest and most versatile method to quantitate nucleic acids in aqueous solution.įigure 1. Recent advances with intercalating fluorescent dyes such as ethidium bromide (6) Hoechst dye 33258 (7) and PicoGreenÒ (8), have improved sensitivity to the picogram level, but are relatively specific for dsDNA limiting their utility in the determination of RNA or oligonucleotide concentrations. These assays all suffer because they inherently destroy the nucleic acid sample. DNA specific assays for hydrolyzed samples (3, 4) with improved sensitivity have also been developed and adapted for cellular DNA synthetic rates (5). These assays are time consuming and relatively insensitive. Early methods utilized measurements of hydrolyzed nucleic acids and by the quantitation of the various components from hydrolysis, such as the phosphorous or the ribose and/or deoxyribose sugar of the backbone, make estimates of total nucleic acid concentrations (1, 2). The common element to all of these diverse molecular biological procedures is the necessity to determine the concentration of a nucleic acid.Īlthough there have been a number of different methodologies developed to quantitate nucleic acids, absorbance at 260 nm remains the de facto standard by which all other methodologies are measured. Quantitation of synthesized oligonucleotides is routinely used to optimize PCR or primer extension experiments. Quantitation of PCR products or plasmid minipreps prior to DNA sequencing has been found to be a critical step in successful DNA sequencing. Likewise, Northern blot analysis requires equal amounts of total or poly A+ RNA be run in each well if comparisons are being made. Southern blot analysis requires that equivalent levels of genomic DNA be loaded in each well. Research investigators from many different fields of study are required to quantitate various nucleic acids in large numbers of samples. Here we describe methodology to quantitate nucleic acids using the BioTek PowerWave™ HT Scanning Microplate Spectrophotometer. D., Senior Scientist, Applications Dept., BioTek Instruments, Inc.Īn essential element of cellular and molecular biology is the ability to quantitate nucleic acids in large numbers of samples at a sensitivity that enables determination of small amounts of sample. This versatile and multi-functional automated system is ideal for research laboratories and core facilities looking to increase productivity while reaping high-quality qualitative and quantitative data to support cell-based research. BioTek Announces New Cytation C10 Confocal Imaging Reader FebruBioTek Instruments, a part of Agilent, is excited to unveil the Cytation C10, an advanced, automated Confocal Imaging Reader.The honor was voted upon by SelectScience members across the world and celebrated at the 2021 Scientists’ Choice Awards online awards ceremony. BioTek's Cytation 7 Cell Imaging Multi-Mode Reader Named ‘Best New Drug Discovery & Development Product’ at Scientists’ Choice Awards BioTek Instruments is pleased to announce that the Cytation 7 Cell Imaging Multi-Mode Reader was awarded ‘Best New Drug Discovery & Development Product’ of 2020.Application flexibility extends to enzyme kinetics, protein stability and aggregation, and projects incorporating loop mediated isothermal amplification (LAMP) assays. Synergy Neo2 and Synergy H1 Now Available with Expanded Temperature Control Range JNow with temperature control up to 70 ✬, BioTek’s Synergy Neo2 Hybrid Multi-Mode Reader and Synergy H1 Hybrid Multi-Mode Reader aid in more assay workflows than ever before.