Biochemistry Science Fair Project
Purification and analysis of plasmid DNA

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Project Information
Title: Purification and analysis of plasmid DNA
Subject: Biochemistry
Grade level: Elementary School - Grades 4-6
Academic Level: Advanced
Project Type: Experimental
Cost: Medium
Awards: 2nd Place, Canada Wide Virtual Science Fair ($50)
Affiliation: Canada Wide Virtual Science Fair
Year: 2013
Materials, Techniques and Concepts: Genomic DNA, plasmid DNA, centrifuge, miniprep, ultraviolet spectrophotometry, agarose gel electrophoresis, E.coli.
Description: The main goal of this project is to verify whether plasmid DNA contains gene sequences. Plasmid DNA was isolated from E. coli using the Miniprep method then the amount of isolated plasmid was quantified using UV spectrophotometry, the plasmid DNA vectors were digested with restriction enzymes and analyzed for the presence of genes using agarose gel electrophoresis.

Plasmid DNA

A plasmid is a small DNA molecule that is physically separate from and can replicate independently of chromosomal DNA within a cell. Most commonly found in bacteria. In nature, plasmids carry genes that may benefit survival of the organism like antibiotic resistance and can frequently be transmitted from one bacterium to another via horizontal gene transfer. Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms.

Plasmids used in genetic engineering are called vectors.

Plasmid preparation is a method used to extract and purify plasmid DNA. Many methods have been developed to purify plasmid DNA from bacteria.

Kits are available from varying manufacturers to purify plasmid DNA, which are named by size of bacterial culture and corresponding plasmid yield. In increasing order, these are the miniprep, midiprep, maxiprep, megaprep, and gigaprep. The plasmid DNA yield will vary depending on the plasmid copy number, type and size, the bacterial strain, the growth conditions, and the kit.

Minipreparation of plasmid DNA is a rapid, small-scale isolation of plasmid DNA from bacteria. It is based on the alkaline lysis method. The extracted plasmid DNA resulting from performing a miniprep is itself often called a "miniprep". Minipreps are used in the process of molecular cloning to analyze bacterial clones. A typical plasmid DNA yield of a miniprep is 50 to 100 g depending on the cell strain.

Plasmids are almost always purified from liquid bacteria cultures, usually E. coli, which have been transformed and isolated. Virtually all plasmid vectors in common encode one or more antibiotic resistance genes as a selectable marker for example ampicillin or kanamycin, which allows bacteria that have been successfully transformed to multiply uninhibited.

The use of plasmids as a technique in molecular biology is supported by bioinformatics software. These programs record the DNA sequence of plasmid vectors, help to predict cut sites of restriction enzymes, and to plan manipulations. Examples of software packages that handle plasmid maps are LabGenius, pDraw32, Geneious and more.

See also:
Plasmid Preparation

Source: Wikipedia (All text is available under the terms of the Creative Commons Attribution-ShareAlike License)

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