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Last update: Mgr. Václav Vopálenský, Ph.D. (25.10.2019)
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Last update: Mgr. Václav Vopálenský, Ph.D. (25.10.2019)
Students will receive protocols with further information during practical exercises. |
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Last update: Mgr. Václav Vopálenský, Ph.D. (25.10.2019)
Students will finish this practocal course by elaborating protocols. |
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Last update: Mgr. Václav Vopálenský, Ph.D. (01.03.2021)
Please note, that the practical course is given in Czech language only. The theoretical part of this course will explain the basic principles of genetic engineering and molecular biology techniques. This theoretical introduction will be used in the followed-up tasks performed in the practical part of this course. Each participant will try the following techniques: 1 / Isolation of plasmid DNA from Escherichia coli cells using the alkaline method followed by analysis of isolated DNA using restriction endonucleases. 2 / PCR amplification of the selected gene and its characterization using agarose electrophoresis. 3 / Chemical transformation of bacteria with DNA vector and determination of transformation efficiency. Analysis of the recombinant protein expression in E. coli culture (protein electrophoresis, a fluorescence analysis of green fluorescent protein in vivo). 4 / In a group of 2-3 students - analysis of proteins using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS - PAAGE). This includes sample preparation and staining of the PAGE gels with Coomassie Brilliant Blue. In case of interest (and if enough time), it will be possible to incorporate protein immunodetection on a solid support matrix, i.e. a western blot. During the whole practice - work with molecular biological databases, annotation of nucleic acid sequences in silico, primers design, etc. The theoretical part of this course will explain the basic principles of genetic engineering and molecular biology techniques. This theoretical introduction will be used in the followed-up tasks performed in the practical part of this course. Each participant will try the following techniques: 1 / Isolation of plasmid DNA from Escherichia coli cells using the alkaline method followed by analysis of isolated DNA using restriction endonucleases. 2 / PCR amplification of the selected gene and its characterization using agarose electrophoresis. 3 / Chemical transformation of bacteria with DNA vector and determination of transformation efficiency. Analysis of the recombinant protein expression in E. coli culture (protein electrophoresis, a fluorescence analysis of green fluorescent protein in vivo). 4 / In a group of 2-3 students - analysis of proteins using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS - PAAGE). This includes sample preparation and staining of the PAGE gels with Coomassie Brilliant Blue. In case of interest (and if enough time), it will be possible to incorporate protein immunodetection on a solid support matrix, i.e. a western blot. During the whole practice - work with molecular biological databases, annotation of nucleic acid sequences in silico, primers design, etc. |