Plasmid Isolation

  • Plasmid isolation is a technique to extract plasmid from plasmid-containing bacteria.
  • Plasmid isolation is a routine task in most cell and molecular biology labs. It is an important step in many procedures such as gene cloning and gene overexpression.
  • A good method of plasmid isolation must be:
    • Rapid
    • Economical
    • Produce plasmid of high quality which can be used for multiple purposes including transfection in cell lines, sequencing, and cloning
  • To isolate the plasmid from the host bacteria, cells are first lysed which leads to the release of plasmid and in subsequent steps, the plasmid is purified from the lysate.
  • There are a number of methods available to lyse bacterial cells. The most common methods are alkaline lysis, boiling lysis, enzymatic lysis, and lysis with detergents.
  • Purification of plasmid from the lysed cells mostly depends on the type of lysis method used to release plasmid in solution. For example, alkaline lysis which completely disrupts the bacterial cells leading to the release of cell components including both plasmid DNA and genomic DNA in a denatured state relies on selective renaturation of only plasmid DNA in a perfect manner at the purification step. On the other hand, boiling lysis selectively releases only plasmid DNA from the bacterial cells.
  • The purified plasmid can be further purified by a number of methods to obtain high-quality plasmids. These methods are centrifugation in gradients of CsCl – ethidium bromide (EtBr), selective precipitation in high salt SDS, extraction with Phenol-chloroform, and hydroxylapatite and ion-exchange chromatography.
  • The following factors can be considered while choosing a method of plasmid 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 the amount of initial culture taken to isolate plasmid DNA.
  • Based on initial culture volume, the 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)
  • The most common methods for plasmid isolation are:
    • Alkaline lysis method
    • Boiling lysis method
  • A typical plasmid isolation method from bacteria includes the following steps
    • Culturing of plasmid-containing bacterial cells in an appropriate growth medium
    • Harvesting of the bacteria
    • Lysis of the bacteria
    • Purification of plasmid DNA

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