Unlike the extraction of genomic DNA, where all DNA is extracted, the isolation of plasmid DNA involves the separation of circular plasmid DNA from bacterial genomic DNA. Furthermore, the majority of the isolated plasmid DNA should remain supercoiled for increased efficiency in downstream applications such as transfection.

There are several methods for isolating plasmid DNA. A typical DNA extraction kit uses some form of alkaline lysis followed by incubation with a DNA binding matrix, washing, and then elution. An optimized DNA extraction kit for plasmids using a modified alkaline lysis method has a number of advantages including:

• Rapid purification of high-quality DNA
• DNA ready for PCR, sequencing, cloning, and other downstream applications
• Contaminants such as bacterial DNA and proteins are removed
• Effective purification from high-protein media such as terrific broth (TB)
• Cost-effective protocol
• No hazardous chemicals such as phenol or chloroform
• No ethanol precipitation required
• Benchtop centrifugation or simple vacuum manifold — no ultracentrifugation

The scale of the plasmid DNA extraction kit used depends on the amounts of starting material and the yield of plasmid DNA that is required. Most kits come in mini, midi, and maxi prep sizes. The majority of applications use the mini kit size, mainly for screening. The midi kit is used for generating sufficient plasmid DNA for multiple manipulations or high-volume applications such as Southern blotting or pulsed field gel electrophoresis.