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Lecturer(s)
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Ohnoutková Ludmila, Ing. Ph.D.
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Vlčko Tomáš, Mgr.
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Course content
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equipment, material, pipetting of small volumes. Isolation of genomic DNA from a leaf according to Edwards et al. (1991). Homogenization with liquid nitrogen. 2) Determination of the concentration and quality of isolated DNA. Spectrometrically, Nanodrop, electrophoretically. Preparation of agarose gel, application of samples, including controls, evaluation. 3) Transformation of chemically competent E. coli cells, DH5?, with an expression vector. Preparation of culture medium, cultivation, E. coli/vector, evaluation of transformation. 4) Isolation of plasmid DNA (Mini-prep, columns). Determination of the concentration and quality of isolated pDNA. Control of isolated pDNA on a gel, specification of pDNA fragments. Comparison with genomic DNA. 5) Transformation of barley, tobacco, etc. plants using Agrobacterium tumefaciens and using biolistics. 6) PCR, principle, design of primers using the Primer3 program for detection of the gene of interest in isolated DNA (Exercise 1). 7) Preparation of PCR reaction mixture including controls. Setting reaction conditions on a thermocycler. Preparation of agarose gel, application of samples. Evaluation using documentation equipment, determination of the presence of the gene of interest in isolated genomic DNA. 8) Colloquium, final test.
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Learning activities and teaching methods
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Laboratory Work
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Learning outcomes
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The aim of the course is to acquire practical and theoretical knowledge in the field of molecular biology with an emphasis on laboratory work. Students will become familiar with the basic equipment and techniques used in a molecular biology laboratory. After completing the course, students will be able to perform basic methods used in molecular biology.
basic methods of molecular biology: DNA isolation, bacterial transformation, plasmid DNA isolation, electrophoretic separation of NK, PCR)
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Prerequisites
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in advance - study instructions, watch recommended educational videos, applications
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Assessment methods and criteria
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Student performance
in advance - study instructions, watch recommended educational videos, applications
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Recommended literature
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Alberts, B. et al. (2014). Molecular Biology of the Cell. Garland Science.
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Brown, T.A. (2016). Gene Cloning and DNA Analysis. WILEY Blackwell.
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Clark, D. Pazdernik, N. (2015). Biotechnology, 2nd Edition. Academic Cell.
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Glick B.R., Pasternak J.J., Patten Ch. (2010). Molecular Biotechnology: Principles and Applications of Recombinant DNA, 4th Edition. ASM Press, USA, 2010. ISBN 978-1-55581-498-4.. Washington DC.
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Šmarda, J. a kol. (2005). Metody molekulární biologie. Brno.
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