Lecture

PCR and Restriction Cloning

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In course

CH-319: Experimental biochemistry and biophysics

A 7-week long (4+8 h) experiment where you plan and construct a fluorescent sensor protein starting from DNA bricks. The protein will be expressed in and purified from E.coli, characterized by biochemical and spectroscopic methods. Your report will be in ...

Description

This lecture covers the genetic engineering and characterization of a FRET-sensor of enzymatic activity, focusing on topics such as PCR cloning, primer design, restriction & ligation, good FRET pairs, electrophoresis, and the design of DNA constructs. The instructor explains the protease cleavage site, the FRET pair, and the conditions for a good FRET sensor. Additionally, the lecture delves into plasmids, restriction endonucleases, DNA ligases, and the polymerase chain reaction process. Practical aspects, such as electrophoresis and DNA sequence analysis, are also discussed, along with the necessary preparations for the laboratory sessions.

Instructor

Rudolf Hovius

Expertise Â•Protein structure, expression & purificationÂ•Molecular interaction & receptor activationÂ•Fluorescence imaging & spectroscopySignalling & sensing Mission => Teaching: Lecture courses, tutorials and practicals in biochemistry, biophysical chemistry of membranes and signalling processes. Participating to the Commission d'Enseignement and Commission de l'Ecole Doctorale of Chemistry and Chemical Engineering.=> Research interests: Protein function and structure, Membrane protein (especially recepto

Official source

Ontological neighbourhood

Biology

Molecular biology: Topics in molecular biology

Related concepts (28)

Gel electrophoresis

Gel electrophoresis is a method for separation and analysis of biomacromolecules (DNA, RNA, proteins, etc.) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF agarose, essentially size independent) and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.

Molecular cloning

Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA.

Plasmid

A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms. In nature, plasmids often carry genes that benefit the survival of the organism and confer selective advantage such as antibiotic resistance.

Agarose gel electrophoresis

Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the two main components of agar. The proteins may be separated by charge and/or size (isoelectric focusing agarose electrophoresis is essentially size independent), and the DNA and RNA fragments by length.

Restriction enzyme

A restriction enzyme, restriction endonuclease, REase, ENase or restrictase is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another.

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