• All About Plasmid DNA Purification

    Plasmid DNA is a molecule that can be double-stranded or circular in shape which can replicate independently of the chromosomal DNA. It plays an important role in the success of some strategies of gene therapy. When used in gene therapy or molecular cloning, it is important that the plasmid DNA is pure to avoid interruptions and repeat of the assays.

    Plasmid DNA purification is very common in research laboratories, especially in preparations for downstream applications, such as PCR, cloning, or transfection experiments. There are several ways that scientists use to purify plasmid DNA, but the two most common methods are the minipreparation and the maximapreparation (bulk preparation.) The type of method to be used in purifying plasmid DNA may depend on the number of plasmid copies, test limit of bacteria, the growth conditions, and the DNA isolation kit being used.

    Minipreparation

    Also known as miniprep, this method of plasmid DNA isolation is based on alkaline lysis and is commonly used for the extraction of small-scale DNA from bacterial cell. In this process, the cell is lysed under alkaline condition, wherein the nucleic acid and protein are modified through acid. Once done, scientists neutralize the solution by adding Potassium Acetate, allowing the chromosomal DNA and protein to selectively precipitate. As a result, the plasmid DNA – given its relatively smaller size – effectively separates from bacteria and proteins.  Miniprep is usually used during molecular cloning to analyze bacterial clones. The typical plasmid yield of miniprep is 20 to 30 µg depending on the cell strain.

    Maxipreparation

    Also known as maxiprep or bulkprep, this method of plasmid DNA isolation is used for much larger bacterial suspension. It involves a scaled-up miniprep and additional purification resulting to a very large amount of extremely pure DNA. The expected DNA yield of maxiprep is 500 to 850 µg.

    Megapreparation

    Also known as megaprep, this method is a scaled-up version of maxiprep used for larger volume of bacterial suspension.  The expected DNA yield of megaprep is 1.5 to 2.5 mg.

    Gigapreparation

    Also known as gigaprep, this aa is the scaled-up version of megaprep for much larger volume of bacterial suspension. The expected DNA yield of gigaprep is 7.5 to 10 mg.

    In plasmid DNA purification, phenol/chloroform is typically added to the bacterial suspension to dissolve and denature proteins like DNase. Researchers use bioinformatics software, such as Lasergene, GeneConstructionKit, and ApE to monitor the process and predict cut sites of the restricted form of plasmid DNA.

    Additional Resources:

    http://en.wikipedia.org/wiki/Plasmid

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