Mastering DNA Isolation

Methods for Isolating DNA from Different Sources

• DNA isolation is an essential step in molecular biology experiments that release DNA, often with other biological molecules, from cells or viruses of interest.
• The method used to isolate DNA depends on the source's structure and composition, and care must be taken to avoid procedures that break DNA.
• The percent of total dry weight that is DNA varies from about 1% in complex mammalian cells to about 50% in bacterial viruses.
• Four procedures are used to isolate DNA: viral, bacterial, plasmid, and yeast/fungal DNA.
• Viral DNA isolation involves mixing an aqueous suspension of virus particles with phenol, which denatures protein molecules and breaks open the protein coat, followed by ethanol precipitation and dissolution in an aqueous buffer solution.
• Bacterial DNA isolation involves treating a bacterial cell suspension with lysozyme to digest the cell wall and then with a detergent such as SDS to disrupt the cell membrane, followed by phenol mixing, ethanol precipitation, and dissolution in a buffered solution containing RNase.
• Plasmid DNA isolation involves incubating the bacterial suspension with lysozyme to digest the cell wall, adding SDS-sodium hydroxide solution to disrupt the cell membrane, and then denaturing the DNA with acid, followed by ethanol precipitation and dissolution in an aqueous buffer solution.
• Yeast and fungal DNA isolation involves breaking down cell walls with enzymes and then following a similar procedure to bacterial DNA isolation, with a focus on protecting RNA from degradation.
• RNA isolation can be a concern due to the potential for RNase degradation, which can be solved by freezing cells in liquid nitrogen and using an acidic solution containing guanidinium thiocyanate, sodium acetate, phenol, and chloroform to isolate RNA from DNA and proteins.
• The upper aqueous phase containing RNA is collected by centrifugation, washed with alcohol, and dissolved in an aqueous buffer solution.
• The isolation procedure for each DNA source must be tailored to the specific cellular or viral DNA source and must consider the source's structures and compositions.
• The isolation procedures for viral, bacterial, plasmid, and yeast/fungal DNA all involve a combination of chemical treatments, mixing, precipitation, and dissolution in an aqueous buffer solution.