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Lecturer(s)
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Gryndler Milan, prof. doc. RNDr. CSc.
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Course content
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1) Introduction: good laboratory practice in molecular genetics, designing the experiments 2) Laboratory exercise 1 - Good laboratory practice 3) Extraction and purification of DNA from microbial cultures (theory), polymerase chain reaction (PCR) use in molecular genetic detection methods (theory), selective DNA primers 4) Laboratory exercise 2 - Extraction and purification of environmental and microbial DNA, PCR 5) Purification of the PCR products, gel electrophoresis, restriction cleavage (theory), gradient gel electrophoresis DGGE and TGGE 6) Laboratory exercise 3 - Purification of the PCR products, gel electrophoresis 7) Methods of the amplicon analysis: RFLP, T-RFLP, RAPD. Ligation of DNA fragments, AFLP 8) Laboratory exercise 4 - Restriction analysis 9) DNA sequencing: Sanger sequencing and high-throughput sequencing methods. The use of sequencing methods in the identification of microorganisms 10) Analysis of sequencing data in microbial ecology 11) Molecular cloning, cloning vectors, heterologous gene expression, expression vectors for E. coli and yeasts. 12) Examples of molecular genetic analysis of microbial communities I. 13) Examples of molecular genetic analysis of microbial communities II.
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Learning activities and teaching methods
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unspecified
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Learning outcomes
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Microbial communities (in soil, in sediments, in composts, etc.) often show high taxonomic diversity and can be studied using various cultivation or molecular methods. The problem connected with application of cultivation methods consists in strong preference for detection of easily culturable microorganisms, while slow growing ones, or these producing small amounts of propagules, are not efficiently detected and unculturable organisms cannot be detected at all. Molecular detection methods are independent on culturability of the microorganisms because they detect DNA from different microbial taxa. The course will provide the basic training in most common molecular methods. Their advantages and limitations will be discussed and practical application protocols will be given.
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Prerequisites
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unspecified
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Assessment methods and criteria
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unspecified
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Recommended literature
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Laboratorní protokoly vytvořené pro potřeby výuky vyučujícím.
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Ruml T. Laboratoře z genového inženýrství. VŠCHT. 1997.
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Vodrážka, Z. Biotechnologie. Academia, Praha,. 1992.
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Vondrejs V. Genové inženýrství I a II. Karolinum, Praha. 2001.
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Zima, T. a kol. Laboratorní diagnostika, Galén, 2002.
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