Basic DNA and RNA Protocols

About this book

An essential core collection of the latest molecular and genetic techniques for cloning, subcloning, sequencing, PCR, protein expression, and much more. Each protocol represents a time-tested, step-by-step recipe that creates an understanding of the procedure, easily reproducible results, and confidence that the procedure will work. The collection includes not only many updated and improved classic techniques, but also a powerful group of advanced methods that point to future progress, among them nonisotopic DNA labeling, silver staining, and automatic sequencing. This excellent bench companion will help those who need to learn for the first time how to conduct research on the molecular biology of nucleic acids or those who need to broaden their competence and laboratory skills. Even highly skilled researchers will find many time-saving techniques.

Contents

Part I. DNA Analysis. The Simultaneous Isolation of RNA and DNA from Tissues and Cultured Cells. Restriction Endonuclease Digestion of DNA. Agarose Gel Electrophoresis. Capillary Blotting of Agarose Gels. Random Primed 32P-Labeling of DNA. Hybridization and Competition Hybridization of Southern Blots. Utilization of DNA Probes with Digoxigenin-Modified Nucleotides in Southern Hybridizations. The Preparation of Fluorescein-Labeled Nucleic Acid Probes and Their Detection Using Alkaline Phosphatase-Catalyzed Dioxetane Chemiluminescence. Monitoring Incorporation of Fluorescein into Nucleic Acid Probes Using a Rapid Labeling Assay. The Preparation of Horseradish Peroxidase Labeled Nucleic Acid Probes and Their Detection Using Enhanced Chemiluminescence. 3'-End Labeling of Oligonucleotides with Fluorescein-11-dUTP and Enhanced Chemiluminescent Detection. The Preparation of Riboprobes. Native Polyacrylamide Gel Electrophoresis. Use of Silver Staining to Detect Nucleic Acids. End-Labeling of DNA Fragments. Part II. RNA Analysis. Northern Blot Analysis. RNA Slot Blotting. The RNase Protection Assay. Primer Extension Analysis of mRNA. S1 Mapping Using Single-Stranded DNA Probes. Nonradioactive In Situ Hybrididzation for Cells and Tissues. Part III. Gene Cloning. In Vitro Packaging of DNA. Construction of Mammalian Genomic Libraries Using l Replacement Vectors. The Production of Double-Stranded Complementary DNA for Use in Making Libraries. Construction of cDNA Libraries. Screening l Libraries. Part IV. Subcloning Methods. Subcloning Strategies and Protocols. Purification of DNA Fragments from Agarose Gels Using Glass Beads. Transformation of E. coli. Transformation of Bacteria by Electroporation. Preparation of Plasmid DNA by Alkaline Lysis2. The Rapid Boiling Method for Small-Scale Preparation of Plasmid DNA. Plasmid Preparations with Diatomaceous Earth. Part V. PCR Techniques. Polymerase Chain Reaction: Basic Protocols. Inverse Polymerase Chain Reaction. Polymerase Chain Reaction with Degenerate Oligonucleotide Primers to Clone Gene Family Members. Cloning PCR Products Using T- Vectors. Direct Radioactive Labeling of Polymerase Chain Reaction Products. A Rapid PCR-Based Colony Screening Protocol for Cloned Inserts. Use of Polymerase Chain Reaction to Screen Phage Libraries. Part VI. DNA Sequencing. Cloning into M13 Bacteriophage Vectors. Ordered Deletions Using Exonuclease III. M13 Phage Growth and Single-Stranded DNA Preparation. Preparation of ssDNA from Phagemid Vectors. A Rapid Plasmid Purification Method for Dideoxy Sequencing. DNA Sequencing Using Sequenase Version 2. 0 T7 DNA Polymerase. Pouring Linear and Buffer-Gradient Sequencing Gels. Electrophoresis of Sequence Reaction Samples. Direct Sequencing of PCR Products. Thermal Cycle Dideoxy DNA Sequencing. Using the Automated DNA Sequencer. Terminal Labeling of DNA for Maxam and Gilbert Sequencing. DNA Sequencing by the Chemical Method. Part VII. Site-Directed Mutagenesis and Protein Synthesis. Site-Directed Mutagenesis of Double-Stranded Plasmids Domain Substitution and Marker Rescue by Comutagenesis of Restriction Enzyme Sites. A Protocol for Site-Directed Mutagenesis Employing a Uracil-Containing Phagemid Template. In Vitro Translation of Messenger RNA in a Rabbit Reticulocyte Lysate Cell-Free System. In Vitro Translation of Messenger RNA in a Wheat Germ Extract Cell-Free System. One-Step Purification of Recombinant Proteins with the 6xHis Tag and Ni-NTA Resin. Index.

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