Cloning Strategies

Content & Learning Objective

In this one-and-a-half-day theory course, basic methods and strategies for the cloning of DNA fragments are explained in detail.

Topics covered include:

• Cloning strategies: successful and efficient cloning
• Vectors: possibilities and choice (standard cloning vectors, eukaryotic expression vectors, inducible systems, etc.)
• Enzymes: isochizomeres, compatible ends, suitable standard enzymes, fill-in and blunt end, dephosphorylation, etc.
• Restriction digest, modification, and preparation
• Ligation: stoichiometry and discussion of different methods
• Transformation: competent bacteria, generation, and efficiency control
• Selection: resistance to antibiotics, blue-white-selection using ß- galactosidase genes, analysis and control of results
• Clone analysis, mini and maxi preps
• DNA quality and purity requirements: gel purification and isolation
• Restriction mapping
• Removal of stop codons
• Tips and tricks for difficult cloning
• Cloning examples
• Trouble shooting

Target Group

Technicians and researchers with previous knowledge of molecular biology who want to get a comprehensive overview of cloning strategies and want to optimize their cloning protocols.

Teacher

Dr. Bettina Füssel studied biology in Cologne and received her PhD in 1996 at the Johann Wolfgang von Goethe University Frankfurt/Main in the field of brain research. Since then, she is working as a principal investigator at the German Center for Cancer Research (DKFZ) in Heidelberg persuing different research projects. Alongside the development of transgenic and knock-out mouse models for different types of disease, her main focus is the intercellular communication between connective tissue and keratinocytes during the development of skin and wound healing.