OceanView Software and Absorbance of Double-stranded DNA

 

OceanView is the flagship software program for Ocean Optics spectrometers. Beyond simple data visualization, OceanView allows users to generate, customize and save spectroscopy projects using its Schematic View function. Saved projects can be reloaded or shared with others. The Schematic View presents each data processing step in a diagram format with icons representing the different devices and actions in your project. In the Schematic, you can view and manipulate the flow of data from your spectrometer through each of your processing steps. Data flow is represented by the use of arrows that connect different nodes. The nodes represent points where data is processed or manipulated.

 

In this project, we calculated DNA concentration from the absorbance measured at 260 nm and the extinction coefficient reported for DNA. DNA and other biological molecules are routinely measured using UV absorbance to obtain critical information on concentration and sample purity.

 

About OceanView Schematic View

 

The Schematic View provides all of the functionality of the OceanView graph view, plus additional capabilities to customize your spectroscopy measurements:

 

• More than 70 algorithm nodes enabling a range of math functions for basic and advanced calculations
• Subrange node to specify a subset of the spectrum or a single wavelength
• Interpolation of spectral data to output data at consistent, evenly spaced intervals
• Scalar view for monitoring a single value such as TEC temperature or an integral or average over a specified wavelength range
• Ability to preview spectra at every step in your process

 

Create, configure and connect schematic nodes to read data from devices, to transform and combine that data through a library of built-in spectroscopic functions, and then to output the results to visual graphs and Microsoft Excel-ready CSV files.

 

Project Description

 

In this project, we calculate the concentration of DNA using Beer’s Law and the extinction coefficient reported for double stranded DNA (dsDNA). To make the measurements, we used an STS-UV microspectrometer (190-650 nm), DH-mini light source, a 1 cm pathlength quartz cuvette in a cuvette holder, and a pair of QP450-1-XSR solarization-resistant patch cords (450 µm diameter, 1 m length).

 

Background

 

Deoxyribonucleic acid (DNA) samples must be well characterized before they are used in various molecular biology techniques. Knowledge of the DNA concentration for a given sample is critical to ensure the use of optimal reagent levels. Purity is assessed because DNA samples are often contaminated with other materials left behind during the purification process.

 

One of the most common methods used to characterize DNA is UV absorbance spectroscopy. The absorbance measured at 260 nm is used to determine DNA concentration and the absorbance measured at 280 nm is used to characterize DNA purity.

 

Project Import Procedure

 

Import the project into OceanView by going to File| Share project| Import. OceanView will guide you through the steps necessary to swap the spectrometer you are using into the project including allowing you to measure new reference and background spectra. We used the standard coefficient for dsDNA measured in a 1 cm pathlength cuvette for this project (50 ug/mL). Note that you will need to update the Schematic if you are working with single stranded DNA (ssDNA) or using a pathlength other than 1 cm.

 

Once the project is loaded, you can use it to determine the DNA concentration for your unknown samples. Additional calculations easily could be added to the OceanView Schematic to report DNA purity.

 

 

References

 

• Clark, W. and K. Christopher. 2000. An introduction to DNA: Spectrophotometry, degradation, and the “Frankengel’ experiment. Pages 81-99, in tested studies for laboratory teaching, Volume 22 (S. J. Karcher, Editor). Proceedings of the 22nd Workshop/Conference of the Association for Biology Laboratory Education (ABLE), 489 pages
• How do I determine the concentration, yield and purity of a DNA sample? Source: Promega.com