genomic dna extraction steps

Genomic DNA, or gDNA, is the chromosomal DNA that encodes the genome of the organism, which includes all the biological information that is transferred to the next generation. It is very large and includes both coding and noncoding regions.

In order to study this, or use it in other applications, it must first be extracted from the cell. This can be accomplished by following a general procedure. First, the walls of the cell membrane must be broken apart. This is referred to as cell lysis, and can be completed a few different ways. It can be done enzymatically using lysozyme, which digests peptidoglycans in the cell wall, and proteinase K, which helps remove contaminants. It can also be achieved mechanically, utilizing a popular bead beating protocol where 0.1 mm glass beads are vortexed with the sample. Mechanical lysis is generally faster and more efficient than enzymatic means. Once lysis has occurred, proteins and other contaminants can be removed by adding detergents and surfactants.

Next, the DNA must be isolated. An ethanol precipitation can be used and enhanced by the addition of sodium acetate, the DNA forming a pellet upon centrifuging. A phenol-chloroform extraction can also be used where phenol denatures the proteins. A couple important points to make are that the sample should be kept at a low temperature to prevent the DNA from degrading. Chemical inhibitors should also be employed to prevent DNAse activity, and a protein precipitation step should be performed to remove the nucleases. These will help maintain the integrity of the DNA for downstream applications.

There are also kits available to streamline this process making it very cost-effective and efficient. They can vary based on the sample type and can yield DNA ready to use in downstream processes. Once isolated, gDNA can be used to make genomic libraries, DNA sequencing, fingerprinting, PCR amplification templates, Southern Blot analysis, SNP analysis, DNA methylation research, and other applications. Again, for most processes, the quality of the data is dependent upon the integrity of the DNA extracted, where great care should be taken to prevent its degradation.

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