Monday, August 3, 2009

Low-volume Quantification of Nucleic Acids using a Monochromator-based Microplate Spectrophotometer

The quantification of nucleic acids is a necessary procedure after isolation from real 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. Spectrophotometery 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 concentrations although for standard 1 cm path length cuvettes, dilution of the sample is typically required. The Epoch™ Multi-Volume Spectrophotometer System is a flexible instrument that provides microvolume quantification of nucleic acids (dsDNA, ssDNA, oligonuleotides) from sub-ng/uL to 4,000 ng/uL without the need for sample dilution. Microanalysis can be performed with as little as 2 uL on 1 – 16 samples simultaneously. The Take3 microplate also allows for 1 cm path length quantification using BioTek’s BioCell accessory or a standard spectrophotometer cuvette. In addition, the Take3 Multi-Volume Plate can be replaced by standard 6 – 384-well microplates for many other applications.
The accuracy of the microvolume quantification of dsDNA is demonstrated in the graph below where standard samples from herring sperm has been measured using undiluted 2 mL samples in the Take3 plate (average of 16 measurements from each of the microwells of the Take3 microplate) and compared to 1 cm pathlength (diluted samples).

Correlation between Take3 and standard cuvette measurements

It is evident that there is excellent correlation between Take3 microvolume determinations and gold standard cuvette-based measurements.
How would you use the plate to take advantage of the 16 well locations? What applications would benefit most from the ability to read low-volume samples, microplates and cuvettes from the same technology?

By: BioTek Instruments

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