Kloniranje fragmenta DNA umnoženog PCR metodom u plazmidni vektor

undergraduate thesis

• Title (english): Cloning of DNA fragment amplified by PCR method into plasmid vector
• Author: Marieta Bagarić
• Mentor: Renata Teparić (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Biochemistry); Zagreb
• Defense date and country: 2017-07, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Kloniranje gena ili dijelova gena u molekularnoj biologiji je skup metoda kojima se željene sekvence iz DNA molekule umnažaju u stanici domaćinu. Eksperimentalni rad započinje umnažanjem željenog fragmenta DNA konvencionalnim metodama poput PCR-a i cijepanjem DNA restrikcijskim enzimima. Nakon toga slijedi ligacija fragmenta DNA (inserta) i lineariziranog plazmidnog vektora kako bi se osiguralo nastajanje plazmida koji se može replicirati u stanici domaćinu te unošenje plazmida u stanicu domaćina u kojoj dolazi do njegovog umnažanja. Na taj način iz jedne rekombinantne DNA nastaje veliki broj istovjetnih molekula DNA što predstavlja samu bit kloniranja. U ovom radu konstruiran je insert Δ246 iz gena SCW4 kvasca Saccharomyces cerevisiae te je ugrađen takozvanom TA-ligacijom u plazmidni vektor pGem-T Easy. Takvim plazmidom su transformirane kompetentne stanice bakterije Escherichia coli. Naposljetku je iz transformiranih stanica izoliran umnoženi plazmid, koji je podvrgnut restrikcijskoj analizi odgovarajućim restrikcijskim enzimima te je rezultat provjeren elektroforetskom analizom restrikcijske smjese.

Abstract (english)

Cloning of genes or gene fragments in molecular biology implies set of methods used to construct desired sequences of DNA and replicate them in host cells. Experimental work primarily sets on creating fragments of DNA by conventional methods including PCR and enzymatic digestion of obtained DNA fragments. Thereafter follows ligation of DNA fragment and linearised vector to ensure formation of plasmid which can replicate itself in the host cell. In this way, out of one recombinant DNA a great number of copies are produced, what is the main goal of cloning. This work is based on the construction of DNA fragment of yeast's Saccharomyces cerevisiae gene SCW4 and it`s ligation into the plasmid vector pGem-T Easy by TA-ligation. This newly created plasmid was introduced into competent cells of Escherichia coli. Cloned plasmid was isolated from transformed cells and the outcome was verified through restriction analisys.

• Keywords: kloniranje; ligacija; transformacija
• Keywords (english): cloning; ligation; transformation
• Language: croatian
• URN:NBN: urn:nbn:hr:159:672521
• Study programme: Title: Biotechnology; Study programme type: university; Study level: undergraduate; Academic / professional title: sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka biotehnologije (sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2017-07-07 11:55:59