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Experiment 3: Isolation, Cloning and Transformation of Plasmid DNA Moksha PatelMadison TomlinsonDr. Martina Hausner BLG 888March 27, 2020
Isolation, Transformation and Cloning of Kanamycin Resistance Gene into pUC18 Plasmid Moksha PatelDepartment of Chemistry and Biology, Ryerson University, 350 Victoria St., Toronto, Ontario,M6B 2K3, Canada[email protected]
1Abstract and KeywordsThe objective of this experiment is to successfully clone a kanamycin resistance gene intothe multiple cloning site of a pUC18 plasmid, and then to transform competent cells with the plasmids. A hypothesis that the kanamycin resistance should be inserted into the pUC18 plasmid through process of isolation, transformation and cloning and growth of white colonies should occur on LB/kan plates was made. Purified plasmids of pU18 and pKan were acquired. Preparation of a 0.7% agarose gel was conducted in which the two plasmids were loaded into thewells. The results obtained from the gel image were inaccurate. Restriction endonucleases HinDIII and BamHI were used to cut the kanamycin resistance gene from the pKan plasmid. Ligation of the kanamycin resistance to the multiple cloning site of the pUC18 plasmid was conducted with DNA ligase. DH5strain of Escherichia coliwas transformed with the plasmids.Indirect and direct methods was used to determine the presence of the kanamycin resistance genein the pUC18 plasmid. For the indirect selection method growth of zero colonies was recorded from the LB/amp/kan plates. For the direct selection method growth of zero colonies was recorded from the LB/kan plates. From the results obtained a conclusion was made that the insertion of the kanamycin resistance gene into the pUC18 plasmid did not occur. Key words:pUC18, transformation, kanamycin resistance gene, -galactosidase, multiple cloning site, E.coli DH5
2IntroductionThe replication process of a certain piece of DNA is called DNA cloning. For the cloning process to being, the DNA piece that is of interest is cut at exact locations using specific endonucleases. A small molecule of DNA that is capable of self-replication and that has restriction sites is chosen which are complementary to the DNA that is of interest. The small molecules chosen are called cloning vectors (plasmid, phage). Using DNA ligase, the piece of DNA that is of interest and the vector are joined together. The combined DNA molecule that is freshly made is called recombinant DNA. By a process of transformation, it is introduced into a host cell in which it can make multiple copies of the host cell and with that it produces multiple copies of the DNA. pUC plasmids are used nowadays most widely as plasmid cloning vectors (Karlovsky, 1987). To manipulate DNA fragments pUC18 vector is most commonly used (Xavier, 2009).