BIO 366 SDSU Introduction for Cloning DNA Lab Report

Description

Introduction:

DNA cloning plays an important role as a laboratory method used in molecular biology. It is simplified as the process of “isolating a DNA sequence of interest for the purposes of making multiple copies of it. This process is implemented when one cuts “a chromosome containing an interesting gene into small pieces, one of which will contain the gene and insert this gene into a plasmid shuttle, or vector, which replicates independently of the original chromosome(Lab Manual, 2020). By performing the procedure that undergoes cloning of DNA, scientists can easily isolate individual genes placed in an orgamsim to undertsnad how each gene functions and further articulate various details within them. This will ultimately help researchers have a closer understanding of our molecular biology today. Such experiments were discovered in 1968 by Arber and Linn who “succeeded in isolating an enzyme, termed a restriction factor, that selectively cut exogenous DNA”(Tirabassi, 2010). Shortly after this, Hamilton Smith expanded onto the study and confirmed Arber and Linn’s discovery by isolating a restriction enzyme from Haemophilus influenza. This major discovery plays a crucial part in the history of biology; it enhanced researchers’ ability to recombine DNA sequences and eventually grow them in different cultures. From this, we were able to familiarize ourselves with Escherichia coli, the most common bacteria used in biological laboratories. 

Recombinant DNA is very much needed when the process of gene cloning is taking place. This procedure is conducted by the usage of a restriction enzyme to cut a single DNA sequence at a particular restriction site. Depending on the restriction enzyme used in the process, a cut can either be blunt or sticky. In this experiment, the restriction enzyme used was a Type II known as Hind III which formed an overhang end also known as a sticky end (Lab Manual, 2020). Once this has been conducted, the usage of a DNA ligase becomes a major part; it used to join the sticky cut end along with the vector. Now, a new recombinant DNA is created and further embedded into a host cell where the process of replication begins. If DNA fragments do not have useful restriction sites for cloning, a polymerase chain reaction (PCR) is used to help further amplify a single piece of a DNA sequence (NCBI, 2000).

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The purpose of these collective experiments was to learn how to clone vectors and insert them into plasmid DNA, as well as identify if they are present by running gel electrophoresis.

In this lab, a plasmid is created through genetic engineering to be resistant to the antibiotic gentamicin. There are two types of antibiotics, one type is bacteriostatic which stops bacterial growth whereas the other type ultimately kills the bacteria. This type is known as bactericidal, one of which is gentamicin. E.Coli cultures do not contain the gentamicin resistance gene. Once the gene for resistance of gentamicin is transferred into the DNA of the E. Coli culture, it can be selected for through the use of gentamicin.

The plasmid temple is first placed in the PCR machine to produce large numbers of copies of DNA. The PCR product and isolated vector plasmid was then digested using HINDIII. Gel electrophoresis was used to separate the PCR digest, digested vector, and undigested vector based on size and charge. The insert and vector were then added to DNA ligase to form the final vector, then electroporated and incubated for the ElectroMAX DH5?-E6 cells to uptake the plasmid. To confirm transduction onto the vector plasmid, the mixture was plated onto an LB agar + gentamicin plate. Only cells with the gentamicin resistant gene would be able to grow and form colonies. Lastly, bioinformatics was used to form a genetic map of the plasmid.

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