What causes DNA to migrate through the agarose gel matrix?
DNA is negatively charged, therefore, when an electric current is applied to the gel, DNA will migrate towards the positively charged electrode. Shorter strands of DNA move more quickly through the gel than longer strands resulting in the fragments being arranged in order of size. They will appear as bands on the gel.
What affect the migration of DNA in gel electrophoresis?
A number of factors can affect the migration of nucleic acids: the dimension of the gel pores (gel concentration), size of DNA being electrophoresed, the voltage used, the ionic strength of the buffer, and the concentration of intercalating dye such as ethidium bromide if used during electrophoresis.
Why is agarose used in gel electrophoresis?
Agarose gel electrophoresis has proven to be an efficient and effective way of separating nucleic acids. Agarose’s high gel strength allows for the handling of low percentage gels for the separation of large DNA fragments.
What influences the migration distance?
The size and shape of a molecule also influence the rate of migration in that the larger the size, the slower the molecule will move in electrophoresis. The viscosity and the pore size in the support media or gels used for electrophoresis influence the rate of migration.
How does agarose concentration affect DNA migration?
The migration rates of DNA molecules in agarose gels are also affected by the composition of the gel. The migration rate of a DNA molecule decreases as the concentration of agarose in the gel increases.
How does agarose gel separate DNA fragments?
To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode.
What is the principle of agarose gel?
Principle of Agarose gel electrophoresis The negatively charged DNA molecules migrate towards the positive charge under the influence of constant current, thus the separation depends on the mass and charge of DNA. The DNA molecules are forced to move through the agarose gel pores.
How does varying the gel concentrations of agarose affect DNA migration through a gel?
The migration rate of a DNA molecule decreases as the concentration of agarose in the gel increases. Researchers commonly adjust the agarose concentration to optimize the resolution of DNA molecules within a particular size range.
On what basis does agarose gel separate molecules?
molecular size
Agarose gel electrophoresis separates the molecules on the basis of molecular size of DNA. Small molecules migrate faster as compared to the larger ones.
How is migration distance measured in gel electrophoresis?
Measure the distance on your picture from the wells to each of the bands in the “ladder,” then divide that distance by the distance traveled by the tracking dye band. This calculation gives you the relative mobility of each band.