Analytical agarose gel electrophoresis
This molecular tool is mainly used to separate and identify DNA fragments and in some cases also to purify certain DNA molecules that can be cut out of the gel after the electrophoresis.
DNA molecules are negatively charged, and by loading them into an agarose gel and applying a current creating a uniform electrical field, these molecules will move across the gel. How far they travel, depends on their electrophoretic mobility. Size, structure and total charge have an impact on how far a molecule will migrate. By comparing the distance the molecules have migrated to a ladder, it is possible to identify what kind of DNA molecule it is or at least its size.
In this experiment, the agarose gel was made by …show more content…
One advantage of this buffer is that DNA can be purified from this gel. Unfortunately the buffer has a minimal buffer capacity, which is a clear disadvantage of this buffer. The buffer serves several important purposes in the electrophoresis. Firstly it carries the current, which is essential for agarose gel electrophoresis. When the current is applied, the water molecules may dissociate into OH- - and H+-ions. DNA molecules are negatively charged and there will therefore be attracting forces between the DNA molecules and and H+-ions. The positively charged hydrogen ions will therefor cancel out the negative charge of the DNA molecules and the DNA molecules will no longer travel across the gel as the current has no impact on them anymore. It is therefore absolutely crucial to have a buffer to prevent this from happening by maintaining a stable pH-value (which should be close to neutral). Another important contribution of the buffer, is to aid in the loading of the gel. The same buffer as the agarose was dissolved in to make the gel, 1xTAE, was poured over the gel until it was covered by 1 mm of the buffer. This is done to increase the density of the gel, so the DNA solutions more easily can be pipetted into the
This molecular tool is mainly used to separate and identify DNA fragments and in some cases also to purify certain DNA molecules that can be cut out of the gel after the electrophoresis.
DNA molecules are negatively charged, and by loading them into an agarose gel and applying a current creating a uniform electrical field, these molecules will move across the gel. How far they travel, depends on their electrophoretic mobility. Size, structure and total charge have an impact on how far a molecule will migrate. By comparing the distance the molecules have migrated to a ladder, it is possible to identify what kind of DNA molecule it is or at least its size.
In this experiment, the agarose gel was made by …show more content…
One advantage of this buffer is that DNA can be purified from this gel. Unfortunately the buffer has a minimal buffer capacity, which is a clear disadvantage of this buffer. The buffer serves several important purposes in the electrophoresis. Firstly it carries the current, which is essential for agarose gel electrophoresis. When the current is applied, the water molecules may dissociate into OH- - and H+-ions. DNA molecules are negatively charged and there will therefore be attracting forces between the DNA molecules and and H+-ions. The positively charged hydrogen ions will therefor cancel out the negative charge of the DNA molecules and the DNA molecules will no longer travel across the gel as the current has no impact on them anymore. It is therefore absolutely crucial to have a buffer to prevent this from happening by maintaining a stable pH-value (which should be close to neutral). Another important contribution of the buffer, is to aid in the loading of the gel. The same buffer as the agarose was dissolved in to make the gel, 1xTAE, was poured over the gel until it was covered by 1 mm of the buffer. This is done to increase the density of the gel, so the DNA solutions more easily can be pipetted into the