Molecular Cloning - MB140C38

• Title: Molecular Cloning
• Czech title: Klonování na úrovni genů
• Guaranteed by: Department of Genetics and Microbiology (31-140)
• Faculty: Faculty of Science
• Actual: from 2025
• Semester: summer
• E-Credits: 3
• Examination process: summer s.:
• Hours per week, examination: summer s.:0/4, C [DS]
• Capacity: 10
• Min. number of students: 6
• 4EU+: no
• Virtual mobility / capacity: no
• State of the course: not taught
• Language: English
• Level: specialized
• Note: enabled for web enrollment
• Guarantor: Mgr. Václav Vopálenský, Ph.D.
• Class: Ultracentrifuga a 3 rotory; Gradientový PCR cycler; Původní předmět

Annotation

English

In this three-day practical course, participants will learn basic molecular cloning techniques from in silico cloning
strategy to the practical molecular cloning of the selected gene into a plasmid-type expression vector with
subsequent verification of the construct using classical molecular biological methods (PCR, restriction
endonuclease digestion or sequencing). Then the course participants will try to express the selected protein in a
bacterial expression system. This part of the practical course includes the electroporation of the constructed
plasmid into the appropriate bacterial strain and the subsequent induction of the expression system to produce
the selected protein, including the subsequent analysis of protein production by PAAGE electrophoresis.

Last update: Lichá Irena, RNDr., CSc. (14.05.2021)

Czech

V tomtočtyřdenním praktickém kurzu se účastníci seznámí s molekulárním klonováním genů od in silico návrhu
klonovací strategie po vložení vybraného genu do expresního vektoru plasmidového typu s následným ověřením
vzniklého konstruktu pomocí klasických molekulárně biologických metod (PCR, štěpení pomocí restrikčních
endonukleáz případně sekvenování). Poté se účastníci kurzu pokusí exprimovat vybraný protein v bakteriálním
expresním systému. Tato část praktického kurzu zahrnuje vnesení vytvořeného plasmidového konstruktu do
zvoleného organismu a následnou indukci systému za účelem produkce vybraného proteinu včetně následné
analýzy pomocí PAAGE elektroforézy.

Last update: Lichá Irena, RNDr., CSc. (27.05.2021)

Learning outcomes

After successfully completion of the course, the student:

·        Defines key concepts and terminology of molecular cloning and recombinant protein expression.

·        Explains the overall molecular cloning workflow from in silico design to experimental verification of recombinant constructs.

·        Identifies appropriate cloning strategies, expression vectors, bacterial host strains, and selection markers.

·        Applies restriction enzyme digestion, DNA ligation, and agarose gel electrophoresis and interprets DNA fragment patterns.

·        Performs isolation and purification of DNA fragments and justifies the importance of DNA quality for downstream applications.

·        Applies bacterial transformation by electroporation and evaluates transformation efficiency.

·        Identifies positive recombinant clones using colony PCR and interprets PCR results.

·        Performs plasmid DNA isolation and explains its role in construct verification and further applications.

·        Explains the principles of regulated recombinant protein expression in bacterial expression systems and applies induction of expression.

·        Describes and applies preparation of bacterial lysates and affinity purification of His-tagged proteins.

·        Applies SDS–PAGE and Western blotting to analyze recombinant protein expression and purification and interprets protein profiles.

·        Evaluates and justifies the suitability, advantages, and limitations of selected molecular cloning and expression methods and summarizes experimental results.

Last update: Vopálenský Václav, Mgr., Ph.D. (18.01.2026)