Optimized Buffers for High-Yield Plasmid DNA Isolation

Many molecular biology techniques rely on the isolation of plasmid DNA from bacterial cultures. Many techniques, such as cloning, sequencing, transfection, and gene therapy rely on the isolation of plasmid DNA.

Plasmid DNA is isolated by first growing a bacterial culture with the plasmid. The cells are lysed in a detergent-containing buffer. Plasmid DNA is then isolated using alcohol-based precipitation or guanidine salts to cause binding of DNA to silica columns.

Genomic DNA is found in eukaryotes and prokaryotes, and is found in the form of chromosomal DNA. It is usually much larger than plasmid DNA. Plasmid DNA is extrachromosomal DNA that is much smaller than genomic DNA and is found only in prokaryotes, like bacteria, and very few eukaryotic organisms.

Separation of plasmid DNA from genomic DNA relies on size, and is often dependent on the right lysis method. This is often done by adding a lysis buffer containing sodium hydroxide and SDS which denatures plasmid and genomic DNA, and then neutralized. Because plasmids are small, they can easily reanneal. However, genomic DNA is too large to reanneal and instead tangles and remains single stranded. Once centrifuged, the tangled genomic DNA will precipitate and form a pellet while the plasmid DNA remains soluble.