Isolation of purified DNA is a required step in many of the core technologies in modern molecular biology. Preparing DNA for downstream analysis requires high purity while maintaining DNA integrity. Isolation of DNA typically follows these steps:
- Cell lysis, or disruption of the cell membrane to allow access to the DNA
- Removal of contaminating lipids, proteins, and RNA
- Precipitation of DNA, typically using isopropanol or ice cold ethanol
This basic procedure is commonly used and quite effective for isolation of both plasmid DNA and chromosomal, genomic DNA. It is possible to selectively isolate extrachromosomal DNA using a technique known as Hirt isolation, which excludes high molecular weight nuclear DNA.
Detecting DNA in solution is a common method for determining DNA concentration and normalizing reactions that involve DNA. There are a handful of DNA detection techniques that are accurate and efficient. Perhaps the most common technique used in molecular biology laboratories is using a spectrophotometer to measure the absorbance of certain wavelengths of light. An absorbance measurement is taken at 260nm and 280nm, and the ratio of the two numbers indicates the purity of the DNA sample. The 260/280 ratio will optimally be at or above 1.8 if the DNA is appropriately pure. DNA can also be analyzed by agarose gel stained with ethidium bromide.