Spectrophotometric analysis and gel electrophoresis are the two main methods used to determine the quality and quantity of extracted nucleic acids.
Spectrophotometric analysis
Spectrophotometric analysis involves using a spectrophotometer to measure the absorption of ultraviolet light by nucleic acids at a specific wavelength of 260 nanometers. This method relies on the principle that DNA and RNA absorb UV light in a characteristic pattern. When a sample containing nucleic acids is exposed to UV light, the amount of light absorbed by the sample is directly proportional to the concentration of nucleic acids present. By measuring the absorbance of the sample at 260 nm, the concentration of nucleic acids can be quantified.
The Beer-Lambert law is then applied to relate the amount of light absorbed to the concentration of nucleic acids in the sample. This method provides a quick and reliable way to determine both the concentration and purity of extracted nucleic acids.
Agarose gel electrophoresis
In the agarose gel electrophoresis technique, DNA concentration is estimated by separating DNA fragments based on their size. In this method, a sample of isolated DNA is loaded into wells of an agarose gel and subjected to an electric field. The DNA backbone, which is negatively charged, migrates through the gel toward the positively charged anode, with smaller fragments moving faster than larger ones. As a result, DNA fragments are separated according to their size, with distinct bands forming in the gel.
By comparing the intensity of these bands to that of known DNA standards loaded onto the gel, the concentration of the sample DNA can be estimated. Additionally, staining the gel with ethidium bromide or SYBR Green allows for visualization of the DNA bands. This method provides a qualitative assessment of DNA concentration and allows for the detection of impurities or degradation products in the sample.