Plasmid isolation

  • Plasmid isolation is a routine task in most cell and molecular biology labs. It is an essential step in many procedures such as gene cloning, DNA sequencing, and transfection.
  • A good method of plasmid isolation must be rapid, economical and produce plasmid of high quality which can be used for multiple purposes including transfection in cell lines, sequencing, and cloning.
  • To isolate plasmid from the host bacteria, cells are first lysed, leading to release of plasmid and in subsequent steps plasmid is purified from the lysate.
  • There are number of methods available to lyse bacterial cells. Most common methods are alkaline lysis, boiling lysis, enzymatic lysis and lysis with detergents.
  • Purification of plasmid from the lysed cells mostly dependent on the type of lysis method used to release plasmid in solution. For example, alkaline lysis which completely disrupt the bacterial cells leading to release of cell components including both plasmid DNA and genomic DNA in denatured state, rely on selective renaturation of only plasmid DNA in a perfect manner at purification step. On the other hand, boiling lysis selectively releases only plasmid DNA from the bacterial cells.
  • Purified plasmid can be further purified by number of methods to obtain high quality of plasmids. These methods are selective precipitation in high salt SDS, centrifugation in gradients of CsCl – ethidium bromide (EtBr), extraction with Phenol-chloroform, and hydroxylapatite chromatography.
  • Following factors can be considered while choosing a method of plasmid isolation isolation……
    • Host strain
    • Plasmid characteristics (copy number and size)
    • Quality and quantity of plasmid
    • Complexity, cost and rapidity of the method
  • The yield of plasmid DNA is governed by two most important parameters – copy number of plasmid and amount of initial culture taken to isolate plasmid DNA. Based on initial culture volume, plasmid isolation method can be termed as…….
    • Miniprep (1 – 5 ml culture volume)
    • Midiprep (25 – 50 ml culture volume)
    • Maxiprep (100 – 500 ml culture volume)
    • Megaprep (1000 – 2500 ml culture volume)
    • Gigaprep (5000 – 10000 ml culture volume)