Sinteza i određivanje osjetljivosti digoksigeninom obilježene probe za hibridizaciju DNA metodom po Southern-u

Bertoša, Rea

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undergraduate thesis

Sinteza i određivanje osjetljivosti digoksigeninom obilježene probe za hibridizaciju DNA metodom po Southern-u

Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology, 2014. urn:nbn:hr:159:008162

Bertoša, Rea

University of Zagreb
Faculty of Food Technology and Biotechnology
Department of Biochemical Engineering
Laboratory for Biology and Microbial Genetics

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APA 6th Edition

Bertoša, R. (2014). Sinteza i određivanje osjetljivosti digoksigeninom obilježene probe za hibridizaciju DNA metodom po Southern-u (Undergraduate thesis). Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology. Retrieved from https://urn.nsk.hr/urn:nbn:hr:159:008162

MLA 8th Edition

Bertoša, Rea. "Sinteza i određivanje osjetljivosti digoksigeninom obilježene probe za hibridizaciju DNA metodom po Southern-u." Undergraduate thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, 2014. https://urn.nsk.hr/urn:nbn:hr:159:008162

Chicago 17th Edition

Harvard

Bertoša, R. (2014). 'Sinteza i određivanje osjetljivosti digoksigeninom obilježene probe za hibridizaciju DNA metodom po Southern-u', Undergraduate thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, accessed 25 April 2024, https://urn.nsk.hr/urn:nbn:hr:159:008162

Vancouver

Bertoša R. Sinteza i određivanje osjetljivosti digoksigeninom obilježene probe za hibridizaciju DNA metodom po Southern-u [Undergraduate thesis]. Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology; 2014 [cited 2024 April 25] Available at: https://urn.nsk.hr/urn:nbn:hr:159:008162

IEEE

R. Bertoša, "Sinteza i određivanje osjetljivosti digoksigeninom obilježene probe za hibridizaciju DNA metodom po Southern-u", Undergraduate thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, Zagreb, 2014. Available at: https://urn.nsk.hr/urn:nbn:hr:159:008162

Cite this item: https://urn.nsk.hr/urn:nbn:hr:159:008162

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Metadata

Title	Sinteza i određivanje osjetljivosti digoksigeninom obilježene probe za hibridizaciju DNA metodom po Southern-u
Title (english)	Synthesis and sensitivity determination of digoxigenin-labelled probe for Southern blotting
Author	Rea Bertoša
Mentor	Ivan-Krešimir Svetec (mentor)
Granter	University of Zagreb Faculty of Food Technology and Biotechnology (Department of Biochemical Engineering) (Laboratory for Biology and Microbial Genetics) Zagreb
Defense date and country	2014-09-11, Croatia
Scientific / art field, discipline and subdiscipline	BIOTECHNICAL SCIENCES Biotechnology
Abstract	Cilj ovog rada bio je sintetizirati te odrediti osjetljivost digoksigeninom (DIG) obilježene probe, komplementarne genu URA3 kvasca Dekkera bruxellensis, koja se koristi pri hibridizaciji DNA po Southern-u. DIG-obilježena proba sintetizirana je lančanom reakcijom polimerazom (PCR), koristeći kao kalup regiju DNA koja sadrži gen URA3, a uspješnost reakcije provjerena je gelelektroforezom. Kako bi se odredila osjetljivost obilježene probe provedena je gel-elektroforeza s različitim masama plazmida koji sadrži gen URA3 kvasca Dekkera bruxellensis. Uzorci razdvojeni elektroforezom preneseni su na nitroceluloznu membranu koristeći vakuum prijenos u lužnatom mediju. Nakon predhibridizacije i hibridizacije denaturiranom DIG-obilježenom probom, detekcija je provedena kolorimetrijskom metodom pomoću BCIP (toluidinska sol 5-brom-4-klor-3’-indolilfosfata) i NBT (nitro-plavi tetrazolijski klorid) koji su korišteni kao supstrat i indikator za alkalnu fosfatazu. Budući da se hibridizacijski signal na membrani pojavio i u uzorku koji je sadržavao svega 1 ng plazmidne DNA može se zaključiti da je sintetizirana proba dovoljno osjetljiva te se može koristiti za analizu genoma kvasca D. bruxellensis.
Abstract (english)	The aim of this work was to synthesize and to determine the sensitivity of digoxigeninlabelled (DIG-labelled) probe, complementary to the URA3 gene from the yeast Dekkera bruxellensis, used for Southern blotting. DIG-labelled probe was synthetized by polymerase chain reaction (PCR), using as a template DNA region that contained URA3 gene, while the success of a PCR reaction was confirmed by gel electrophoresis. To determine the sensitivity of the probe, gel electrophoresis with various masses of the plasmid DNA, containing URA3 gene from the yeast D. bruxellensis was performed. Samples separated by gel electrophoresis were transferred to a nitrocellulose membrane using alkaline vacuum transfer. DNA was prehybridized and hybridized with DIG-labelled probe while the detection was done by colorimetric method using BCIP (toluidine-salt of 5-bromo-4-chloro- 3-indolyl-phosphate) and NBT (nitro blue tetrazolium chloride), as a substrate and indicator for the alkaline phosphatase, respectively. Since hybridization signal was obtained even with the sample containing only 1 ng of the plasmid DNA, we can conclude that newly synthesised probe is sensitive enough and can be used for yeast D. bruxellensis genome analysis.
Keywords
Keywords (english)
Language	croatian
URN:NBN	urn:nbn:hr:159:008162
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	Access restricted to students and staff of home institution
Terms of use
Repository	Repository of the Faculty of Food Technology and Biotechnology
Created on	2017-04-26 11:46:12

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