Table of Contents
What is the purpose of buffer in agarose gel?
The function of TBE and TAE buffer is to allow nucleic acids to move through the agarose matrix. Therefore, the agarose gel must be completely submerged in the buffer. In addition, the TBE or TAE buffer maintains the pH and ion concentration during electrophoresis.
What causes DNA to move in the agarose gel?
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 is agarose gel composed of?
Agarose is a high-molecular-weight polysaccharide extracted from the cell walls of certain marine red algae. Chemically, it is a copolymer of 1,3-linked β-d-galactose and 1,4-linked 3,6-anhydro-α-l-galactose.
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.
What is the role of boric acid in TBE buffer?
The borate ions in Tris-borate-EDTA (TBE) buffers interact with DNA to form highly charged DNA-borate complexes, which are stable both in free solution and in polyacrylamide gels.
What is the role of electrophoresis buffer?
High-quality buffers are an important part of electrophoresis. They allow a current to be carried through the sample while resisting pH changes in the overall solution. These buffers facilitate the separation of the samples into readable gels.
How does polyacrylamide gel electrophoresis work?
As an electric current is applied proteins migrate through the gel to the positive electrode as they have a negative charge. Each molecule moves at a different rate based on its molecular weight – small molecules move more rapidly through the gel than larger ones. Migration is usually faster at higher voltages.
Why does DNA separate in gel electrophoresis?
Based on their size and charge, the molecules will travel through the gel in different directions or at different speeds, allowing them to be separated from one another. All DNA molecules have the same amount of charge per mass. Because of this, gel electrophoresis of DNA fragments separates them based on size only.
How is DNA visualized on agarose gel?
DNA is visualized by including in the gel an intercalating dye, ethidium bromide. DNA fragments take up the dye as they migrate through the gel. Illumination with ultraviolet light causes the intercalated dye to fluoresce with a pale pink colour.
Why are DNA fragments separated?
Gel electrophoresis is a technique used to separate DNA fragments (or other macromolecules, such as RNA and proteins) based on their size and charge. All DNA molecules have the same amount of charge per mass. Because of this, gel electrophoresis of DNA fragments separates them based on size only.
What are the roles of boric acid and EDTA?
TBE or Tris/Borate/EDTA, is a buffer solution containing a mixture of Tris base, boric acid and EDTA. As these ions are necessary co-factors for many enzymes, including contaminant nucleases, the role of the EDTA is to protect the nucleic acids against enzymatic degradation.