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Low Volume Nucleic Acid Quantification using A Multi-Volume Microplate Spectrophotometer SystemDownload
September 11, 2009
Authors: Peter Brescia, Lenore Buehrer and Gary Barush, BioTek Instruments, Inc.
The quantification of nucleic acids is a necessary procedure after isolation from samples such as tissues, cells or body fluids. Downstream applications include PCR, RT-PCR, sequencing, restriction digestions and ligations. All these applications involve enzymatic reactions where efficiency is dependent on the relative concentrations of nucleic acid, enzyme and other reactants, hence the need for quantification. Amounts of nucleic acid isolated from most kits can range anywhere from ng to μg and are typically eluted in 10 to 100 μL volumes. Nucleic acid concentrations can range from sub ng/μL to thousands of ng/μL. Spectrophotometry is a very popular method for nucleic acid quantification as it is a simple, accurate and non-destructive method for the measurement of nucleic acid over much of the range of concentrations described above. For standard 1 cm pathlength cuvettes, dilution of the sample is typically required for nucleic acid concentrations above about 100 – 200 ng/μL to avoid Beer’s Law non-linearity issues at high optical density. Here we describe the Epoch™ Multi-Volume Spectrophotometer System that allows the user to rapidly measure from 1 -16 samples in 2 μL volumes without any need for dilution. The unique Take3™ plate included in the System also provides the capability to read BioTek’s BioCell™ or any standard 1 cm pathlength cuvette. Finally, the Epoch can be used as a standard monochromator-based microplate spectrophotometer by replacement of the Take3™ plate with any standard 6- to 384-well density microplate for many other applications.