Low Volume Nucleic Acid Quantification using a Multi-Volume Spectrophotometer System
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Low Volume Nucleic Acid Quantification using a Multi-Volume Spectrophotometer System
Authored by: Peter Brescia and Peter Banks, BioTek Instruments, Inc. Winooski, VT
Micro-volume quantification allows for rapid assessment of nucleic acid concentrations. The Epoch™ Multi-Volume Spectrophotometer System provides the ability to measure a wide range of samples from 2 μL to mL volumes using the Take3™ Multi-Volume Plate. Here we demonstrate the utility of Epoch and Take3 for dsDNA quantification and compare accuracy to gold standard techniques.
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Introduction
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 microgram to nanogram levels 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 a wide range of concentrations. 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 nonlinearity issues at high optical density.
Here we describe a spectrophotometer equipped with a specialized accessory to allow the user to rapidly measure a variety of sample volumes and formats.
Materials and Methods
The Epoch Multi-Volume Spectrophotometer System (BioTek Instruments, Winooski; Figure 1) consists of the Epoch Microplate Spectrophotometer and Take3 Multi-Volume Plate. The Epoch’s UV/Vis optical system with continuous wavelength selection can perform endpoint reading, spectral scanning, kinetic and well area scanning measurements in standard 6- to 384-well microplates.
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| Figure 1. The Epoch Multi-Volume Spectrophotometer System incorporating the Epoch Spectrophotometer and Take3 Multi-Volume Plate. |
The Take3 Multi-Volume Plate (Figure 2) has a standard SBS footprint with 16 microspots arranged as columns akin to columns 2 and 3 of a 96-well microplate. Two microliter samples can be pipetted into individual microspots, or a multichannel pipette may be used to load eight samples simultaneously. The Take3 also allows measurement of 1 cm pathlengths using BioTek’s BioCell (for a fixed vertical pathlength) or any standard spectrophotometric cuvette.
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| Figure 2. Take3 plate with 16 microspots (2 μL) for micro-volume measurements. The plate can also accommodate 2 BioCells, and a standard 1 cm pathlength cuvette. |
All double-stranded DNA (dsDNA) standards were created by serial dilution of a concentrated herring sperm dsDNA stock in TE buffer (10 mm TRIS, 1 mm EDTA, pH=7.0). Take3 micro-volume data was obtained with undiluted standard samples. Each standard concentration was loaded five times at each microspot location on the Take3 plate using an 8-channel manual pipettor and absorbances read at 260, 280 and 320 nm. BioCell data was acquired using either undiluted or, for higher concentration samples, a 20-fold dilution of standard in TE. All sample measurements were background corrected using a TE buffer blank at 260 nm. All concentrations depicted are based on a 1 cm pathlength and 50 ng/μL/OD.
Reproducibility across 16 Microspots in the Take3 Plate
Figure 3 demonstrates the DNA concentrations obtained using the micro-volume feature of the Take3 Multi-Volume Plate to measure a 2050 ng/μL dsDNA standard. This represents the typical upper end of the DNA concentration range obtained from DNA plasmid isolation kits such as Invitrogen’s PureLike HiPure maxi kit. The optical density obtained for this undiluted dsDNA sample averaged 1.8 ODs for each of the micro-volume microspots. With a 1 cm pathlength, this undiluted sample would produce a theoretical OD of approximately 36, assuming the Beer’s law was still obeyed. The %CV across the 16 microspots was 1.8%. The % relative standard deviation across the 16 microspots is 1.8%.
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| Figure 3. dsDNA concentration determinations of a 2050 ng/μL herring sperm dsDNA sample using all 16 microspots of a Take3 plate. |
Accuracy using 16 Microspots of a Take3 Plate
The accuracy of micro-volume quantification was determined across a broad range of herring sperm dsDNA concentrations. The replicate measurements from the 16 microspots were used to determine average concentrations and the standard deviation was used as vertical error bars in Figure 4, below.
It is apparent from the slope of the graph in Figure 4 that there is a 2% difference in accuracy across the broad range of dsDNA concentrations between undiluted micro-volume determinations and 1 cm pathlength determinations made with the BioCell.
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| Figure 4. Accuracy of micro-volume quantification of dsDNA relative to measurements made at 1 cm pathlength with BioCell accessory. Both micro-volume and BioCell measurements are made with the Take3 Multi-Volume Plate. |
Conclusions
The Epoch Multi-Volume Spectrophotometer System serves as a micro-volume quantitative spectrophotometer; a cuvette-based spectrophotometer for highly accurate absorbance measurements; and finally as a monochromator-based scanning microplate absorbance reader for performing ELISAs and other colorimetric assays.
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