Gel Purification

introduction

In molecular biology, gel extraction or gel isolation is a technique used to isolate the desired fragment of untouched DNA from an agarose gel following agarose gel electrophoresis. After extraction, the desired fragments can be mixed, precipitated, and enzymatically ligated in several simple steps.

This process, which is commonly performed on plasmids, is fundamental to basic genetic engineering. After running DNA samples on an agarose gel, the extraction involves four essential steps: identifying the desired fragments, isolating the corresponding bands, separating the DNA from those bands, and removing salts and contaminants. To start, UV light is applied to the gel to illuminate all DNA stained with ethidium bromide. Care must be taken to avoid exposing the DNA to mutagenic radiation for longer than necessary. The target band is marked with a blade and physically removed. The excised piece of gel should contain the desired DNA within it.

Gel Purification Procedure

Gel extraction kits are available from several major biotechnology manufacturers, The protocols in these kits generally indicate that the gel should first be melted at 50 degrees Celsius, followed by the extraction of the sample from the gel using an appropriate chaotropic agent, 70% alcohol, and water.

Applications of Gel Purification

Enzymatic Digestion: This is a crucial step in cloning, as it is essential for the subsequent steps such as ligation. Therefore, it is very important that the vector and plasmid used for ligation are pure.

Purification of Vector Digestion: After digesting the vector, since the small fragment of the vector is over 40 base pairs, direct use of a silica column is not feasible. Thus, gel extraction is employed.

It can be confidently stated that the most important step in cloning is the ligation phase, and many preliminary tasks we perform, such as PCR of our target gene, are all aimed at enhancing the efficiency of the ligation step. In the ligation reaction, the materials used include the digested gene.