Agarose Gel Electrophoresis
Since the discovery of the four classes of macromolecules (proteins, lipids, carbohydrates, and nucleic acids), researchers and biochemists have fine-tuned methods that reveal structural information about each of them. Different structural properties determine different functions and chemical properties, and scientists have developed intricate techniques to observe, quantify, and isolate molecules based on their aforementioned properties. Agarose gel electrophoresis, the most widely used technique in separating strands of DNA, is used to visualize and isolate strands of nucleic acids based primarily on length. The technique can be used on a mixed assortment of DNA molecules to yield information concerning the length …show more content…
It is important to use a calibrated DNA ladder to interpret results. A DNA ladder contains reference bands along the gel representing known distances that DNA molecules of specific known lengths travel, and is usually provided by manufacturers. Depending on the size of DNA molecules, different concentrations of agarose may be used. For resolution of smaller bands, a higher percentage concentration of agarose gel should be used (approximately 2% gel). Conversely, a small percentage concentration of agarose gel is sufficient in resolving large bands from one another (approximately 0.7% gel). It is also crucial for the preservation of DNA molecules to add a buffer such as TAE or TBE to the agarose matrix. Fluctuations in pH can cause denaturation of DNA molecules. When heating the agarose gel, it is important not to over-boil the mixture as some buffer may be lost to evaporation at high …show more content…
Ethidium bromide is able to situate itself in the major grooves of DNA molecules. Because ethidium bromide binds to DNA molecules in a dose-dependent manner, the intensity of each band under UV correlates directly to the amount of DNA present in solution. Ultraviolet light is applied post-electrophoresis to visualize the bands; however, the gel should not be subjected to UV longer than 30-45 seconds if the ultimate goal is to purify molecules. UV light damages DNA molecules and is a known cause of mutagenesis. Recent studies have shown that ethidium bromide is mostly likely carcinogenic. This has caused a number of laboratories to move away from its use, instead preferring to use dyes such as crystal violet and methyl blue that do not require exposure to ultraviolet light. Still, ethidium bromide is cheap, relatively easy to use, and less sensitive than other dyes, so most researchers continue using it carefully. Ethidium bromide actually delays migration of DNA by approximately 15% due to its positive charge, but most manufactured DNA ladders account for
Since the discovery of the four classes of macromolecules (proteins, lipids, carbohydrates, and nucleic acids), researchers and biochemists have fine-tuned methods that reveal structural information about each of them. Different structural properties determine different functions and chemical properties, and scientists have developed intricate techniques to observe, quantify, and isolate molecules based on their aforementioned properties. Agarose gel electrophoresis, the most widely used technique in separating strands of DNA, is used to visualize and isolate strands of nucleic acids based primarily on length. The technique can be used on a mixed assortment of DNA molecules to yield information concerning the length …show more content…
It is important to use a calibrated DNA ladder to interpret results. A DNA ladder contains reference bands along the gel representing known distances that DNA molecules of specific known lengths travel, and is usually provided by manufacturers. Depending on the size of DNA molecules, different concentrations of agarose may be used. For resolution of smaller bands, a higher percentage concentration of agarose gel should be used (approximately 2% gel). Conversely, a small percentage concentration of agarose gel is sufficient in resolving large bands from one another (approximately 0.7% gel). It is also crucial for the preservation of DNA molecules to add a buffer such as TAE or TBE to the agarose matrix. Fluctuations in pH can cause denaturation of DNA molecules. When heating the agarose gel, it is important not to over-boil the mixture as some buffer may be lost to evaporation at high …show more content…
Ethidium bromide is able to situate itself in the major grooves of DNA molecules. Because ethidium bromide binds to DNA molecules in a dose-dependent manner, the intensity of each band under UV correlates directly to the amount of DNA present in solution. Ultraviolet light is applied post-electrophoresis to visualize the bands; however, the gel should not be subjected to UV longer than 30-45 seconds if the ultimate goal is to purify molecules. UV light damages DNA molecules and is a known cause of mutagenesis. Recent studies have shown that ethidium bromide is mostly likely carcinogenic. This has caused a number of laboratories to move away from its use, instead preferring to use dyes such as crystal violet and methyl blue that do not require exposure to ultraviolet light. Still, ethidium bromide is cheap, relatively easy to use, and less sensitive than other dyes, so most researchers continue using it carefully. Ethidium bromide actually delays migration of DNA by approximately 15% due to its positive charge, but most manufactured DNA ladders account for