Basic Cloning Procedures

Experience in laboratory experiments in molecular biology is important to students, researchers and practioners in the biolo- gical and medical sciences. This book is designed to introduce the reader to basic methods used in the isolation, cloning and analysis of genetic material. The text includes theory and fundamental methods in suffi- cient experimental detail which have been successfully used in our studies of viral genome and eukaryotic gene regulation. Spe- cific protocols together with troubleshooting tips are given for RT-PCR amplification, gene cloning, hybridization analysis and sequencing of nucleic acids, PCR-based site-specific mutagene- sis, analysis of protein DNA-specific interaction, cell-free protein synthesis and product electrophoretic and immunological analy- sis. The methods chosen have been approved in several interna- tional Molecular Biology Courses and without doubt can be used as a manual in basic molecular studies of DNA and RNA. The editor is grateful to all the contributors for their coopera- tion and would like to express his thanks to the publishers, espe- cially Dr. J. Lindenborn from Springer-Verlag, for their coopera- tion, patience and assistance during the preparation of this book. Riga, Latvia V. BERZINS Contents Chapter 1 cDNA Synthesis and Cloning ...1 JURIS STEINBERGS AND ALEKSANDER TSIMANIS Chapter 2 DNA Sequencing...22 ...ERIKS JANKEVICS Chapter 3 RNA In Vitro Synthesis by Phage T7 DNA-Dependent RNA Polymerase...43 ...ELITA AVOTA AND NORMUNDS LICIS Chapter 4 PCR-Based Site-Specific Mutagenesis...53 ...JANIS KLOVINS AND VALDIS BERZINS Chapter 5 Analysis of Specific Protein-DNA Interactions...68 . .