In this article we will discuss about the Concentration of DNA samples and its measurement.
Concentration of DNA Samples:
Organic extraction often results in a very high concentration of DNA that does not need to be concentrated any further. Other purification methods give more dilute solutions and therefore, it is important to consider methods for increasing the DNA concentration.
The most frequently used method of increasing concentration is ethanol precipitation. In the presence of monovalent cationic salt (NaCl) and at a temperature of -20 degrees centigrade or less, absolute ethanol efficiently precipitates polymeric nucleic acids.
With a thick solution of DNA the ethanol can be layered on top of the sample, causing molecules to precipitate at the interface. A spectacular trick is to push a glass rod through the ethanol into the DNA solution. When the rod is removed, DNA molecules adhere and can be pulled out of the solution in the form of long fibres.
Alternatively, if ethanol is mixed with a dilute DNA solution, the precipitate can be collected by centrifugation.
It is very crucial to know exactly how much DNA is present in a solution while carrying out gene cloning experiment. DNA concentrations can be accurately measured by ultraviolet (UV) absorbance spectrophotometry.
The amount of UV radiation absorbed by a solution of DNA is directly proportional to the amount of DNA in the sample. Usually absorbance is measured at 260 nm, at which wavelength an absorbance of (A260) of 1.0 corresponds to 50 micrograms of double stranded DNA per ml.
UV absorbance can also be used to check the purity of a DNA preparations. With a pure sample of DNA the ratio of the absorbance’s at 260 nm (A260/A280) is 1.8. Ratio of less than 1.8 indicates that the preparation is contaminated, either with protein or with phenol.