Polymerase Chain Reaction (PCR), Restriction Enzyme Digestion, Ligation and DNA gel electrophoresis

7 Pages • Essays / Projects • Year Uploaded: 2021

Genetic engineering is the process of manipulating the genetic material of an organism — often to include the DNA from a foreign organism. It is a crucial step in the design of fermentation and bioprocess because only well-designed expression system of the host cell can ensure the constant production of the desirable products. Prior to perform the transformation assay, students will first amplify a gene of interest via PCR. A plasmid will be cleaved by restriction enzyme at specific sites. Following that the gene of interest will be introduced to the plasmid vector followed by annealation of ligase, a process called ligation. In this practical, the plasmid pGLO will be used as the vector for the incorporation of the gene of interest. The gene of interest in this study is a gene that encodes the green fluorescent protein in a bioluminescent jellyfish, Aequorea victoria. This plasmid contains a multiple cloning sites that permit insertion or deletion of the gene of interest. Students will practice on cutting out the GFP gene from the plasmid DNA and reintroduce the (PCR amplified) GFP gene by ligation. DNA gel electrophoresis is run to observe if the plasmid ligation is successful.

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