Question

1- How do you determine the concentration of your purified DNA sample? What is spectrophotometry? What are the basic principles of spectrophotometry?

2- What is the purpose of determining the A260/A280 ratio?

3- At what wavelength of light does DNA maximally absorb?

4- Explain Gel electrophoresis. How does it work? How is it useful?

Answer 1

QUESTION 1, 2 and 3

Nucleic acids efficiently absorb ultraviolet light due to the presence of nitrogenous aromatic bases along the DNA strands. UV absorption of DNA is a characteristic of the molecule, which is used efficiently to determine its concentration. Each of the bases has its own unique absorption spectrum and therefore contributes differently to the total UV absorption property of a DNA molecule.

The nucleic acids have a maximum absorption at a wavelength of 260nm. At this length, therefore, the absorption is proportional to the concentration.

The concentration and purity of a DNA sample can be determined by spectrophotometry based on the absorbance capacity of a compound present in a solution at a given wavelength. In this way the concentration of the DNA sample is calculated taking into account the absorbance value obtained at a wavelength of 260 nm. While the ratio of absorbances A260/280 and A260/230 are used to evaluate the purity of the samples.

The A260/280 ratio is very stable and it has been reported that a pure DNA has a ratio between 1.8-2.0. A DNA of acceptable purity must have at least one A260/280 ratio > 1.6. On the other hand, a value A260/280 < 1.6 indicates a possible contamination by aromatic compounds such as phenols and proteins. A radio A260/280 > 2.1 could be due to the presence of RNA in the sample.

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