This is not just a cookbook for real-time quantitative PCR (qPCR). Admittedly, there are lots of recipes from distinguished contributors and Bustin has attempted to collect, sift through and rationalize the vast amount of information that is available on this subject. And yes, this book was conceived as a comprehensive hands-on manual to allow both the novice researcher and the expert to set up and carry out qPCR assays from scratch.
However, this book also sets out to explain as many features of qPCR as possible, provide alternative viewpoints and methods and, perhaps most importantly, aims to stimulate the researcher into generating, interpreting and publishing data that are reproducible, reliable and biologically meaningful.
In this book, leading scientists from academia as well as biotech and pharma companies introduce the revolutionary concept of designing RNA and DNA oligonucleotides with novel functions by in vitro selection. These functions comprise high affinity binding (aptamers), catalytic activity (ribozymes and deoxyribozymes) or combinations of binding and catalytic properties (aptazymes).
Basic concepts and technologies describing in detail how these functional oligonucleotides can be identified are presented. Numerous examples demonstrate the versatility of in vitro selected oligonucleotides. Special emphasis has been put on a section that shows the broad applicability of aptamers, e. g. in target validation, for analytics, or as new therapeutics. This first overview in the field is of prime interest for a broad audience of scientists both in academia and in industry who wish to expand their knowledge on the potential of new oligonucleotide functions and their applications.
This resource presents protocols for isolating a gene, cloning and characterizing it, expressing its encoded protein, and purifying and characterizing the protein′s physical properties. It includes background and procedures and is structured around 20 experiments that demonstrate how to prepare, manipulate, and analyze plasmids, produce fusion proteins in bacteria, and purify these proteins based on chemical properties or substrate affinities.
It describes topics such as the use of antibodies and techniques developed to transform their structures, and approaches designed to manipulate structure and functions of proteins and nucleic acids.
In Volume 2 of Essential Molecular Biology, procedures for preparing gene libraries and identifying genes are described, together with methods for studying the structure of a cloned gene and the way it is expressed in the cell.
Protein expression is an increasingly important tool for research on gene function. What is needed is not just a lab manual providing established methods as well as the latest state-of-the-art protocols, but also clear advice on what expression system to choose when. This book covers expression across a broad range of systems, including the following. *Baculovirus expression vectors, *CHO cells, * E. coli, *HEK293-EBNA1 cells, * Lactococcus lactis, * S. cerevisiae, *transfected insect cells, * Pichia pastoris, *mammalian cells using BacMam viruses, *lentiviral vectors, *wheat germ cell-free system. The book takes the reader through how to make an informed choice of appropriate system, taking into account the protein target, the time involved, the ultimate use of the expressed protein, and the laboratory equipment required. In addition, the book describes the optimisation of expression strategies, expression engineering using ribosome display, and how to select protein variants with improved expression.
Highly skilled investigators in gene targeting and mouse genetics describe their best techniques for the design of targeting constructs and for the analysis of the mouse phenotype. These include embryo transplantation, in vitro embryonic stem cell differentiation, creation of aggregation chimeras, mouse pathology, embryo cryopreservation, and transplantation. State-of-the-art and highly practical, it constitutes an invaluable source of readily reproducible techniques for genetic researchers today.
Gene transfer to animal cells was first achieved more than 30 years ago. Since then, transformation technology has developed rapidly, resulting in a multitude of techniques for cell transformation and the creation of transgenic animals. As with any expanding technology, it becomes difficult to keep track of all the developments and to find a concise and comprehensive source of information that explains all the underlying principles. This book describes the principles behind gene transfer technologies, how gene expression is controlled in animal cells and how advanced strategies can be used to add, exchange or delete sequences from animal genomes in a conditional manner. The final chapter provides an overview of all the applications of animal cell transformation.
This revised and updated edition of a recognized classic emphasizes tissue and cell in situ hybridization methods. Among the new techniques detailed are PNA probes for viral diagnostics, plant in situ hybridization, cell proliferation detection, and quantitation of in situ hybridization. There are also cutting-edge techniques for tissue microarrays, expanded embryology–developmental gene detection, and expanded cell culture. Derivative techniques presented include identification of transplanted cells, histones, nick-end labeling for apoptosis, the use of peptide nucleic acid probes, and in situ hybridization of plant specimens. The protocols include step-by-step laboratory instructions, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
The major histocompatibility complex is the subject of much research in the immunology field. There is a great deal of interest in MHC proteins and their function as antigen presenting molecules, and many immunology laboratories are investigating biochemical and genetic techniques to study these molecules. The identification of peptide transporter genes and the elution of peptides from MHC molecules illustrate how rapidly our understanding of the MHC locus has advanced. These volumes bring together the technologies, which make these advances possible. It explains in detail the process such as peptide translocation into the ER, the application of mass spectrometry to the analysis of peptides bound to MHC, and signal detection via MHC class II molecules, which lead to either activation or cell death.
This edition is a massive revision of the previous edition. It is the classic in-depth text reference in cell biology, yet puts the latest hot topics sensibly in context - including genomics, protein structure, array technology, stem cells and genetic diseases. It incorporates and emphasized new genomic data. It not only sets forth the current understanding of cell biology, but also explores the intriguing implications and possibilities of that which remains unknown. It contains over 1,600 illustrations, electron micrographs, and photographs, of which over 1,000 are originally conceived by the authors. "Cell Biology Interactive", a CD-ROM, is packaged with every copy.
This sixth edition of James D. Watson′s classic textbook Molecular Biology of the Gene has been thoroughly revised and updated. Accessible to anyone interested in molecular biology and genetics, the book provides a historical basis for the field, concise descriptions of fundamental chemical concepts, a comprehensive survey of genome maintenance and expression, and a discussion of standard techniques and model organisms commonly used in molecular biology studies. It includes all new chapters on the regulatory RNAs and genomics and systems biology. The book has an accompanying Web site (www.aw-bc.com/watson/), which contains interactive tutorials, animations, and critical-thinking exercises designed to help students explore and visualize complex concepts.
In this new edition, authors Joe Sambrook and David Russell have completely updated the book, revising every protocol and adding a mass of new material, to broaden its scope and maintain its unbeatable value for studies in genetics, molecular cell biology, developmental biology, microbiology, neuroscience, and immunology. As in earlier editions, this is the only manual that explains how to achieve success in cloning and provides a wealth of information about why techniques work, how they were first developed, and how they have evolved. It includes 240 laboratory protocols in DNA science in which over 35% were created especially for this edition, along with coverage of bioinformatics and DNA microarrays.
Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Molecular Cloning is the new gold standard-the one indispensable molecular biology laboratory manual and reference source. Highlights of the new edition: • Extensive new content: 12 entirely new chapters are devoted to the most exciting current research strategies, including epigenetic analysis, RNA interference, genome sequencing, and bioinformatics • Expanded scope: the nucleic-acid-based techniques selected for inclusion have promoted recent advances in gene transfer, protein expression, RNA analysis, and expression of cloned genes • Classic content: 10 original core chapters have been updated to reflect developments and innovations in standard techniques and to introduce new cutting-edge protocols • Easy-to-follow format: the previous editions′ renowned attention to detail and accuracy are fully retained • Essential appendices: an up-to-date collection of reagents, vectors, media, detection systems, and commonly used techniques are included • Expanded authorship: chapters and protocols have been specifically commissioned from renowned experts at leading institutions
A methods manual for studying all aspects of NO and its metabolism. Readily reproducible techniques form a core of effective experimental methods for researchers new to the field and serve as a ready reference for experienced investigators. Cloning and expression of NO synthase isoforms, measurement of NO synthase activity, direct and indirect quantitation of NO, and the role of NO in DNA damage and apoptosis are all covered. In addition to stepwise instructions, each protocol also summarizes pros and cons and describes pitfalls and troubleshooting.
This edition update and expands Bruce White′s best-selling "PCR Protocols" (1997) with the newest procedures for DNA cloning and mutagenesis. Here the researcher will find readily reproducible methods for all the major aspects of PCR use, including PCR optimization, computer programs for PCR primer design and analysis, and novel variations for cloning genes of special characteristics or origin, with emphasis on long PCR and GC-rich template amplification. Powerful applications of PCR in library construction and sublibrary generation and screening are presented.
This book covers gene analysis at the level of restriction fragment polymorphism, point mutation, and sequence analysis. Methods are given for using different source materials: cloned DNA, genomic DNA, RNA, nucleic acid from archive material, and PCR products themselves.
A detailed practical guide for laboratory use aimed at those who wish to use efficient and reliable protocols in their work and at researchers checking the validity and interpretation of published data. These molecular cytogenetic methods can be used to characterize and locate nucleic acid sequences within the cell. It is presented in a user-friendly, open format for easy access to information with an extensive troubleshooting guide.
With a variety of detection chemistries, an increasing number of platforms, multiple choices for analytical methods and the jargon emerging along with these developments, real-time PCR is facing the risk of becoming an intimidating method, especially for beginners. Real-time PCR provides the basics, explains how they are exploited to run a real-time PCR assay, how the assays are run and where these assays are informative in real life. It addresses the most practical aspects of the techniques with the emphasis on ′how to do it in the laboratory′.
This update presents techniques for generation and analysis of transgenic animals, with emphasis on manipulation of the mammalian genome. It concentrates on creation and maintenance of genetically modified murine strains, providing access to the germline by conventional pronuclear injection, and by retroviral and adenoviral infection. Emphasis is given to the generation, maintenance, and manipulation of embryonic stem cell lineages, with protocols for constitutive and conditional gene targeting. Chapters include techniques for cryopreservation of male and female germlines and for generation of transgenic sheep by nuclear transfer.
Using Whole Genome Amplification (WGA) methods, it is possible to create microgram quantities of DNA starting with as little as one nanogram of genomic DNA and in some cases even a single eukaryotic or bacterial cell. The implementation of such WGA methods provides an ample supply of DNA for large-scale genetic studies. This title provides a comprehensive overview of the field and will be welcomed by all researchers looking to take advantage of the latest developments.
This volume, part of the Advances in Molecular Biology series, presents detailed protocols and trouble-shooting advice on the yeast two-hybrid system, one of the most powerful and versatile methods for characterizing a protein′s functions. Topics include: characterizing hormone/receptor complexes, identifying peptide ligands, how to identify mutations that disrupt an interaction, how to construct an activation domain hybrid library, and how to dissect the cell cycle and other complex genetic networks.