Recombinant DNA Technology. Social Sciences. Lab Report

Recombinant DNA Technology
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Recombinant DNA Technology
Introduction
DNA cloning refers to a technique used by molecular biologists to prepare several identical copies of a gene or a DNA fragment. During gene cloning, a target piece of DNA is inserted into a plasmid or a circular piece of DNA, which is then introduced into a bacterial genome through a process known as transformation (Alberts et al, 2002). The bacteria that take up the plasmid are then selected by using antibiotic markers. The bacteria with the right plasmid are used to prepare more plasmid DNA or can be used to make proteins or induced to express a gene of interest. The process of gene cloning requires two kinds of enzymes: restriction enzymes and DNA ligases. However, in most advanced molecular biology laboratories, a common approach used to make several copies of a DNA fragment is polymerase chain reaction (PCR). PCR uses DNA polymerase enzymes to produce new DNA strands from the existing DNA templates (National Center for Biotechnology Information & U.S. National Library of Medicine, 2017). In this project, a different approach known as cell-based cloning will be used to make DNA copies using E.coli cells. Cell-based cloning ensures that large quantities of DNA are obtained in a limited amount of time. This technique also facilitates the identification and repair of mutations and thus produces a small number of mutants.
There are three steps that will be followed in this project: recombinant DNA creation, E.coli cells transformation, and selection using antibiotic markers. The first step in this experiment will be to create a recombinant DNA or plasmid. This circular piece of DNA is often found in bacteria which has the capacity to replicate independently outside a chromosomal DNA. In this experiment, pBluescript or pBluescript KS plasmid will be used. These plasmids will be prepared by the laboratory technician before the commencement of the project. pBluescript allows the selection of the transformed bacteria because it possesses ampicillin resistance property. The plas...