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.

How This Product Works

The Kit relies on an alkaline lysis to free the plasmid DNA from the cell, leaving behind the E. coli chromosomal DNA trapped in the cell wall debris. After the solution is cleared of cell debris and chromosomal DNA, the supernatant is retained and passed to the spin Filter Tube. The nucleic acid binds specifically to the surface of glass fibers in the presence of chaotropic salt (guanidine HCl). Since the binding process is specific for nucleic acids, the bound plasmid DNA is purified from salts, proteins and other cellular impurities by washing steps followed by elution in low-salt buffer or water.

Applications

• PCR
• Sequencing
• Cloning
• In vitro transcription
• Restriction enzyme digests
• Random primed labeling