explain how mobility and size correlate and all the factors that allow for the m

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There are many factors that influence the migration of DNA fragments in the agarose gel. 1) Size of the DNA 2) Concentration of agarose in the gel 3) Conformation of the DNA fragments 4) Voltage applied to the gel 5) Concentration of EtBr The size of the DNA fragments plays a crucial role in affecting the migration of the DNA molecules in the agarose gel. It is well observed that separation of DNA fragments in the gel is achieved due to resistance to their movement caused by the gel matrix. Therefore, this resistance is the driving force, which causes the DNA molecules to migrate differentially. While moving towards the anode, the larger DNA molecules experience most of the frictional force. They encompass the most difficulty while passing through the pores of the gel. Therefore, the mobility of larger DNA fragments is relatively slow as well as retarded during gel electrophoresis. Moreover, the distance they migrate from the well is reasonably less. On the other hand, the DNA molecules of smaller size experience the least frictional force. Literally, they encounter less difficulty while passing through the pores of the gel. Hence, the mobility of small DNA fragments is comparatively fast as well as unhindered during gel electrophoresis. In addition, the distance they migrate from the well is logically more. Therefore, in order to find out the size of the desired DNA fragment in the gel, the samples are loaded along with DNA size markers (a set of known size of different DNA fragments), commonly know as DNA ladders. Now-a-days various ranges of DNA ladders are available commercially for different ranges of DNA fragments. For example, 50bp ladder (range 50bp-3Kb), 100bp ladder (range 100bp-1Kb), 1Kb ladder (range 250bp-10Kb), etc. Concentration of agarose in the gel has a significant effect on the mobility of the DNA fragments in the gel. It has been observed that higher concentrations of agarose cause the gel matrix to become dense. As a result of this, the pore size of the gel decreases. Small pore size of the gel exerts more resistance to the migration of the DNA fragments. Hence, this leads to the slower migration of fragments in the gel. On the other hand, lower concentrations of agarose cause the gel matrix to be thin. As a result of this, the pore size of the gel increases. Large pore size of the gel exerts less resistance to the migration of the DNA fragments. Therefore, this leads to the faster migration of the fragments in the gel. Based on these observations, the concentration of agarose in the gel is adjusted according to the size of the fragment to be analyzed in the gel. For example, if the DNA molecule is plasmid of 10Kb or higher then concentration of agarose in the gel is adjusted around 0.7%. However, if the DNA fragment is a PCR product of 300bp or less then the concentration of agarose in the gel is adjusted around 1.2-1.4% or higher. For the DNA fragments falling under the range of 10Kb-300bp, the concentration of agarose is adjusted to 1%. Motility of DNA fragments migrating in the gel is also influenced by the conformation of the DNA molecule. Generally there are three conformations of a DNA fragment: Supercoiled or covalently closed circular DNA, nicked or open circular DNA, and linear DNA. It is a well observed fact that plasmids exists as circular supercoiled molecules (i.e. ends of the plasmid DNA are not free rather joined to form the circle) in the bacterial cell. Topoisomerases are the key enzymes involved in the procedure of supercoiling of the DNA fragments. The phenomenon occurs when the double stranded DNA molecule is coiled upon itself after the replication process. In order to maintain the supercoiled conformation of both the DNA strands, it is mandatory for the strands to be intact. That is, the whole length of the strand should be intact and there should be no nick or cuts. That is why; this conformation is also referred as the covalently closed-circular (ccc) DNA. Due to its supercoiled nature, the DNA fragments become smaller in size and hence experience less frictional resistance from the gel. This results in the migration of this conformation of DNA to be faster than other conformations. If one of the double strands of the circular supercoiled DNA fragment is broken due to nick or cut, the double helix reverts to its normal i.e. uncoiled or relaxed state. As a result of this the plasmid DNA changes its conformation which is called as open-circular (oc) or relaxed. This conformational change results in the increase in size of the DNA molecule and hence it experiences more frictional resistance from the gel. As a result of this, the migration of open-circular DNA is slower than other conformations. When both the strands of the supercoiled DNA fragment is broken due to nick or cut, the circular double helix becomes linear. Because of this conformation it migrates at normal speed (see figure) i.e. migrates according to its size. Therefore, migrations of different conformations of DNA fragment follow the order: supercoiled > linear > open circular. This difference in migration due to conformational change is the main reason to get three bands when a pure plasmid is loaded on the gel. 4) Voltage applied to the gel It is a well known fact that applied voltage is the major driving force behind the migration of the DNA fragments in the gel. Therefore, it is quite obvious to assume its role in the migration of DNA fragments. Generally, the applied voltage is kept in the range of 5-8V/cm, where distance between the electrodes is measured in centimeters (cm). Migration of the DNA fragments is directly proportional to the applied voltage. For example, increase in the applied voltage results in the increase in the migration of the DNA fragments in the gel. However, decrease of the voltage causes slower migration of the DNA molecules. The applied voltage depends upon the purpose of gel electrophoresis. If the gel electrophoresis is done for southern hybridization then the applied voltage is adjusted to 1-3V/cm. This results in the slower migration of fragments in the gel and hence better resolution. If electrophoresis is done for routine analysis of DNA fragments, then the applied voltage is adjusted to 5-8V/cm. Moreover, if electrophoresis is done for the purification of the fragments from the gel, then the applied voltage is adjusted to 3-5V/cm In addition, the applied voltage also depends on the size of the DNA fragments. If the fragments are smaller in size then electrophoresis is done at higher voltage. This results in the better resolution and less diffusion of the fragments in the gel. However, for fragments of larger size, the applied voltage is decreased. Moreover, the concentration of agarose has also effect on the applied voltage. For the proper resolution of fragments in the gel having higher agarose concentration, the applied voltage is decreased. However, the applied voltage is increased if the concentration of agarose in the gel is low. 5) Concentration of EtBr You know that EtBr is an intercalating agent used for the visualization of DNA fragments in the gel. It intercalates between the stacked base-pairs of the DNA double helix. As a result of this, the migration of DNA fragments is affected. Intercalation of EtBr into the DNA double helix increases the size of the DNA fragments by a fair margin. This leads to the slower migration of the fragments. It has been observed that rate of migration of DNA fragments is slowed by approximately 15% by the addition of EtBr. Because of this, for southern blotting rather than adding EtBr to the gels, these gels are stained after the migration of the DNA fragments. This improves the resolution of the gel, and fragments migrate according to their respective size. These are the major factors affecting the migration of the DNA fragments in the agrose gel.

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