Springer Protocols Handbooks – serie

Research in the field of molecular biology has progressed at a fascinating rate in recent years. Much of this progress results from the development of new laboratory techniques that allow very precise fractionation and analysis of nucleic acids and proteins, as well as the construction of recom­ binant DNA molecules that can then be cloned and expressed in host cells. Progress has been so rapid that there has been a shortfall in the training of appropriately qualified staff. Many existing laboratory workers require retraining, and many educational institutions have had difficulty incor­ porating the new molecular biology techniques into their teaching programs. Although there are several manuals currently available that describe laboratory techniques in molecular biology, they are principally written for the indivi­ dual research worker and are not intended for use in the design of practical classes for students. The aim of this book is to provide just such a series of pro­ tocols forthe teaching of practical molecular biology. The idea arose following the success of several Workshops in Molecular Biology, organized and taught by staff in the Biol­ ogy Department of the Hatfield Polytechnic. Gradually, the protocols used in the workshops have been incorporated into the Hatfield undergraduate and postgraduate teaching pro­ grams and have now been collected together to form a book.

There can be no doubt that some ofthe most spectacular advances made in science over the past few decades have been in the isolation, analysis, and manipulation of nucleicacids. Thishas ledtoamuchgreaterunderstandingofmechanismsandprocesses across many fields of bioscience, such as biochemistry, microbiology, physiology, pharmacology, and the medical sciences to name a few. It has also led to the growth of the biotechnology industry, which seeks to develop and commercialize many ofthese important processes and methods. Much ofthis has come about because ofthe devel- opment of numerous molecular biology and genetic manipulation techniques. The discovery of restriction enzymes and the development of cloning vectors in the early 1970sopenedthedoortowaysofisolatingandmanipulatingnucleic acidsthathadnever been thought possible. Gene probe labeling and hybridization were developed and refined toprovidepowerfulmethodsofanalysis. These-togetherwiththedevelopment of DNA sequencing methods, protein engineering techniques, and PeR-have all continued to contribute substantially to the understandingofbiological processes at the molecular level.Theprotocols for these importantmethods are the focus ofThe Nucleic AcidProtocols Handbook, whose aim is to provide a comprehensive set oftechniques in onevolume thatwill enable the isolation, analysis, and manipulationofnucleic acids to be readily undertaken. The NucleicAcidProtocols Handbook is divided into 10 parts; within each there are approximately 10chapters. The first fourpartsfollow oneanotherlogically: nucleic acid extraction (Part I), basic separation and analysisofDNA (II), through probe design and labeling (III), and RNA analysis techniques (IV). The following three sections deal with gene libraryconstruction andscreening(V), DNA sequencing (VI), andthe polymerase chain reaction (VII).

The contributors to this manual are uniformly from those labora- ries routinely using microwave technology to facilitate their processing methods in the various fields of microscopy. The methods and results these authors describe are the tangible evidence that microwaves can be used routinely as the basis for improved sample processing for micr- copy applications. These applications include complete sample proce- ing protocols for light and electron microscopy, decalcification, and immunocytochemistry. The overall time savings, ease of use, and qu- ity of results serve as justification for using microwaves in the labo- tory. The question as to whether there is a "microwave effect" is alluded to, but not discussed in any great detail. When the term microwave technology is used, it is generic and intended to mean equipment designed for laboratory versus household use. Microwave Techniques and Protocols is designed for anyone with a background and experience in sample processing for immunocytoch- istry, decalcification, light microscopy, or electron microscopy, and clearly demonstrates that microwave technology has a place in today's laboratory. Richard T. Giberson, Richard S.Demaree Jr. , REFERENCES Giberson, R. T. Demaree, R. S. , and (1995) Microwave fixation: understa- ing the variables to achieve rapid reproducible results. Microsc Res Tech 32:246-254. Giberson, R. T. , Demaree, R. S. , Jr. , and Nordhausen, R. W. (1997) Four-hour processing of clinical/diagnostic specimens for electron microscopy using microwave technique. J Vet Diagn Invest 9:61-67. Jr. VhD Ms vi Preface ...v Contributors ...

This book provides a comprehensive overview of the major biochemical analytical techniques, with detailed descriptions of the instrumentation and applications. The contributions, which each focus on a specific technique, are based on a thorough review and analysis of the current literature as well as the authors’ experiences in the lab. Divided into nine parts, the book provides insights into basic separation techniques like sedimentation, filtration and centrifugation, as well as analytical techniques such as spectrophotometry, chromatography, electrophoresis, immuno-techniques, radioactivity and microscopy.

This book provides a comprehensive overview of the major biochemical analytical techniques, with detailed descriptions of the instrumentation and applications. The contributions, which each focus on a specific technique, are based on a thorough review and analysis of the current literature as well as the authors’ experiences in the lab. Divided into nine parts, the book provides insights into basic separation techniques like sedimentation, filtration and centrifugation, as well as analytical techniques such as spectrophotometry, chromatography, electrophoresis, immuno-techniques, radioactivity and microscopy.

This volume details methods and protocols necessary to further the study of insect immunity. Chapters guide readers through up-to-date genomic and transcriptomic approaches, insect samples for proteomic analysis, hemocytes in Drosophila, cellular response in Lepidoptera, insect AMPs, manipulate the composition of mosquito microbiota, viral infections in insects, infections by entomopathogenic nematodes, immune response following oral infections, and protocols to to monitor the effect of septic infections with human pathogens using B. mori as a model. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Immunity in Insects aims to ensure successful resultsin the further study of this vital field.

This volume details methods and protocols necessary to further the study of insect immunity. Chapters guide readers through up-to-date genomic and transcriptomic approaches, insect samples for proteomic analysis, hemocytes in Drosophila, cellular response in Lepidoptera, insect AMPs, manipulate the composition of mosquito microbiota, viral infections in insects, infections by entomopathogenic nematodes, immune response following oral infections, and protocols to to monitor the effect of septic infections with human pathogens using B. mori as a model. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Immunity in Insects aims to ensure successful resultsin the further study of this vital field.

The book presents hands-on protocols for conventional and advanced forensic DNA fingerprinting experiments. It includes manual, semi-automatic, and advanced automatic techniques for DNA extraction from different biological samples.It also discusses various qualitative and quantitative approaches for the assessment of extracted forensic DNA. It contains protocols for the amplification of short tandem repeat markers (STRs) for the amplification-based target enrichment of the forensic samples.Further, it examines genotyping of the STR loci through capillary electrophoresis and includes real-world case studies where forensic DNA analysis has been used in the criminal and civil disputes. The book concludes by presenting technological developments in the field of DNA forensic analysis. Suitable for beginners, it is a key reference resource on a wide variety of DNA profiling techniques and applications.

The book presents hands-on protocols for conventional and advanced forensic DNA fingerprinting experiments. It includes manual, semi-automatic, and advanced automatic techniques for DNA extraction from different biological samples.It also discusses various qualitative and quantitative approaches for the assessment of extracted forensic DNA. It contains protocols for the amplification of short tandem repeat markers (STRs) for the amplification-based target enrichment of the forensic samples.Further, it examines genotyping of the STR loci through capillary electrophoresis and includes real-world case studies where forensic DNA analysis has been used in the criminal and civil disputes. The book concludes by presenting technological developments in the field of DNA forensic analysis. Suitable for beginners, it is a key reference resource on a wide variety of DNA profiling techniques and applications.

This book provides detailed information on methodologies used in biological, serological and nucleic acid based assays for the detection, diagnosis and management of plant viruses. The content is divided into six main parts, the first of which presents techniques used in the biological characterization and transmission of viruses, while Part II covers purification and techniques concerning the physico-chemical properties of viruses. In turn, Part III focuses on in vitro expression, production of polyclonal and monoclonal antibodies; and on various serological assays including precipitin tests, ELISA, blot immunoassays, immunosorbent electron microscopy and lateral flow immunoassays. Part IV addresses the isolation of DNA and RNA from plants and nucleic acid based assays such as dot-blot, polymerase chain reaction, real-time PCR, loop-mediated isothermal amplification, rolling circle amplification, recombinase polymerase amplification, and next-generation sequencing approaches. Part V discusses cloning, sequencing, sequence analysis and the production of infectious clones, while the last part (Part VI) provides biotechnological approaches for the management of viruses. Given its scope, the book will be a valuable resource for every laboratory, student and teacher, and for everyone interested in plant virology, plant pathology, plant biology and molecular biology, offering them a practical manual on various aspects of plant viruses.

This book provides detailed information on methodologies used in biological, serological and nucleic acid based assays for the detection, diagnosis and management of plant viruses. The content is divided into six main parts, the first of which presents techniques used in the biological characterization and transmission of viruses, while Part II covers purification and techniques concerning the physico-chemical properties of viruses. In turn, Part III focuses on in vitro expression, production of polyclonal and monoclonal antibodies; and on various serological assays including precipitin tests, ELISA, blot immunoassays, immunosorbent electron microscopy and lateral flow immunoassays. Part IV addresses the isolation of DNA and RNA from plants and nucleic acid based assays such as dot-blot, polymerase chain reaction, real-time PCR, loop-mediated isothermal amplification, rolling circle amplification, recombinase polymerase amplification, and next-generation sequencing approaches. Part V discusses cloning, sequencing, sequence analysis and the production of infectious clones, while the last part (Part VI) provides biotechnological approaches for the management of viruses. Given its scope, the book will be a valuable resource for every laboratory, student and teacher, and for everyone interested in plant virology, plant pathology, plant biology and molecular biology, offering them a practical manual on various aspects of plant viruses.

This manual provides insights into the theory and practical aspects of several biotechnological and biochemical techniques for plants, protozoa, nematodes, insects and fishes, as well as human samples. The book also covers bioinformatics tools. The manual is an inclusive compilation, explaining techniques for microbial cultures, their diagnostics, DNA barcoding, microscopic techniques, blood analysis, parasite diagnostics through copro-antigens, enzyme analysis with enzyme kinetics, gene expression analysis, in-vivo protein visualization in live animals, geno-toxicity assays, quantification of micro RNAs and LncRNAs in tissue sections, the use of droplet PCR, and in-silico analysis. It provides step-by-step descriptions and details of each methodology, together with the final outcomes and inferences, in a simple and lucid manner, easily reproducible even for beginners. The broad range of techniques covered makes this volume extremely useful in understanding the principles of biotechniques, and simple applications for practical courses.

This manual provides insights into the theory and practical aspects of several biotechnological and biochemical techniques for plants, protozoa, nematodes, insects and fishes, as well as human samples. The book also covers bioinformatics tools. The manual is an inclusive compilation, explaining techniques for microbial cultures, their diagnostics, DNA barcoding, microscopic techniques, blood analysis, parasite diagnostics through copro-antigens, enzyme analysis with enzyme kinetics, gene expression analysis, in-vivo protein visualization in live animals, geno-toxicity assays, quantification of micro RNAs and LncRNAs in tissue sections, the use of droplet PCR, and in-silico analysis. It provides step-by-step descriptions and details of each methodology, together with the final outcomes and inferences, in a simple and lucid manner, easily reproducible even for beginners. The broad range of techniques covered makes this volume extremely useful in understanding the principles of biotechniques, and simple applications for practical courses.

This volume details the fundamentals of the CRISPR-Cas system, and its protocols illustrate advances in CRISPR-Cas techniques for efficient genome editing. Introductory chapters provide a wide horizon of CRISPR/Cas-based methods and applications. Additional chapters guide readers through HDR-mediated editing, sgRNA design, the step-by-step procedure of multiplex adenine base editing experiments in rice, generating mutants for rice, wheat, Brachypodium, Barley, Flax, and Phytophthora, visual screening of mutants, gene deletion (knock-out), tagging (knock-in) in mammalian cells, the cloning-free (DNA-free) technique, cell-penetrating peptides, generating a genome-edited banana, and nuclear genome editing of Chlamydomonas employing CRISPR-Cpf1 combined with a single-stranded DNA (ssODN) repair template. Authoritative and cutting-edge, CRISPR-Cas Methods aims to assist researchers who are new to the field and are aiming to learn how best to adopt this technology for a particular organism.

This volume details the fundamentals of the CRISPR-Cas system, and its protocols illustrate advances in CRISPR-Cas techniques for efficient genome editing. Introductory chapters provide a wide horizon of CRISPR/Cas-based methods and applications. Additional chapters guide readers through HDR-mediated editing, sgRNA design, the step-by-step procedure of multiplex adenine base editing experiments in rice, generating mutants for rice, wheat, Brachypodium, Barley, Flax, and Phytophthora, visual screening of mutants, gene deletion (knock-out), tagging (knock-in) in mammalian cells, the cloning-free (DNA-free) technique, cell-penetrating peptides, generating a genome-edited banana, and nuclear genome editing of Chlamydomonas employing CRISPR-Cpf1 combined with a single-stranded DNA (ssODN) repair template. Authoritative and cutting-edge, CRISPR-Cas Methods aims to assist researchers who are new to the field and are aiming to learn how best to adopt this technology for a particular organism.

This book provides a timely overview of analytical tools and methodological approaches for studying membrane lipids. It outlines the ground-breaking advances that have been made over the last two decades in high-throughput lipidomics, and in studying lipid-protein interactions, signalling pathways and the regulation of lipid metabolism.This user-friendly laboratory handbook is an ideal companion for membrane biologists, researchers, students, and clinicians alike. It is also well suited for teaching biochemistry, microbiology and biotechnology courses, making it a must-have for everyone whose work involves lipid research.

This book provides a timely overview of analytical tools and methodological approaches for studying membrane lipids. It outlines the ground-breaking advances that have been made over the last two decades in high-throughput lipidomics, and in studying lipid-protein interactions, signalling pathways and the regulation of lipid metabolism.This user-friendly laboratory handbook is an ideal companion for membrane biologists, researchers, students, and clinicians alike. It is also well suited for teaching biochemistry, microbiology and biotechnology courses, making it a must-have for everyone whose work involves lipid research.

This thorough book aims to present the methods that have enabled the success of peptides and proteins in a wide variety of applications. It opens with a section on chemical tools applied to the production or engineering of peptides and proteins, and concludes with a collection of chapters on biological approaches used to engineer structure and function in peptides and proteins. As a book in the Springer Protocols Handbooks series, chapters include the kind of detailed descriptions and tips necessary for successful results in practice. Authoritative and practical, Peptide and Protein Engineering: From Concepts to Biotechnological Applications will be of great use to scientists in academia and industry seeking a better understanding of the emerging principles and methodologies in peptide and protein engineering.

This thorough book aims to present the methods that have enabled the success of peptides and proteins in a wide variety of applications. It opens with a section on chemical tools applied to the production or engineering of peptides and proteins, and concludes with a collection of chapters on biological approaches used to engineer structure and function in peptides and proteins. As a book in the Springer Protocols Handbooks series, chapters include the kind of detailed descriptions and tips necessary for successful results in practice. Authoritative and practical, Peptide and Protein Engineering: From Concepts to Biotechnological Applications will be of great use to scientists in academia and industry seeking a better understanding of the emerging principles and methodologies in peptide and protein engineering.

This comprehensive laboratory manual describes the various protocols involved in Actinobacterial research. The content is divided into fifteen major sections, each of which is further divided into sub-sections describing the respective aim, principles, materials & methods, protocol, expected results and diagrams. Readers will find essential protocols for e.g. sample collection, isolation, characterization, analysis, profiling and evaluation of Actinobacteria for various applications. Gathering all relevant protocols concerning Actinobacteria, and written by a team of experienced Actinobacterial researchers, it is the first book of its kind.

This comprehensive laboratory manual describes the various protocols involved in Actinobacterial research. The content is divided into fifteen major sections, each of which is further divided into sub-sections describing the respective aim, principles, materials & methods, protocol, expected results and diagrams. Readers will find essential protocols for e.g. sample collection, isolation, characterization, analysis, profiling and evaluation of Actinobacteria for various applications. Gathering all relevant protocols concerning Actinobacteria, and written by a team of experienced Actinobacterial researchers, it is the first book of its kind.

This manual details the techniques involved in the study of plant microbe interactions (PMI). Covering a wide range of basic and advanced techniques associated with research on biological nitrogen fixation, microbe-mediated plant nutrient use efficiency, the biological control of plant diseases and pests such as nematodes, it will appeal to postgraduate students, research scholars and postdoctoral fellows, as well as teachers from various fields, including pathology, entomology and agronomy.It consists of five broad sections featuring different units. Information panels at the beginning of each unit present essential knowledge as well as advances in a particular topic. The manual can also serve as a textbook for undergraduate courses like Techniques for Plant-Microbe Interactions; Biological Control of Plant Diseases; and Nutrient Use Efficiency. Providing basic insights and working protocols from all related disciplines, this unique laboratory manual is a valuable resource for researchers interested in investigating PMI.

This manual details the techniques involved in the study of plant microbe interactions (PMI). Covering a wide range of basic and advanced techniques associated with research on biological nitrogen fixation, microbe-mediated plant nutrient use efficiency, the biological control of plant diseases and pests such as nematodes, it will appeal to postgraduate students, research scholars and postdoctoral fellows, as well as teachers from various fields, including pathology, entomology and agronomy.It consists of five broad sections featuring different units. Information panels at the beginning of each unit present essential knowledge as well as advances in a particular topic. The manual can also serve as a textbook for undergraduate courses like Techniques for Plant-Microbe Interactions; Biological Control of Plant Diseases; and Nutrient Use Efficiency. Providing basic insights and working protocols from all related disciplines, this unique laboratory manual is a valuable resource for researchers interested in investigating PMI.

Understanding the functional roles of DNA modifications relies on the accurate detection, quantification, and mapping of DNA modifications. Methods for deciphering DNA modifications have substantially improved over the last several years, which greatly revolutionize the field of DNA modifications. In addition to DNA cytosine methylation (5-methylcytosine, 5mC), the best-characterized epigenetic modification, many new modifications have been discovered to present in DNA in recent years.  This book provides a comprehensive overview of available techniques and methods together with detailed step-by-step protocols for experimental procedures required to successfully perform analysis on various types of DNA modifications, including 5mC, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 5-carboxycytosine (5caC), 5-hydroxymethyluracil (5hmU), 5-formyluracil (5fU), N6-methyladenine (6mA), β-D-glucosyl-5-hydroxymethyluracil (base J) and 8-oxo-7,8-dihydroguanine (OG). This laboratory manual is a valuable source for biochemists and molecular biologists from different fields who wish to investigate DNA modifications.

Understanding the functional roles of DNA modifications relies on the accurate detection, quantification, and mapping of DNA modifications. Methods for deciphering DNA modifications have substantially improved over the last several years, which greatly revolutionize the field of DNA modifications. In addition to DNA cytosine methylation (5-methylcytosine, 5mC), the best-characterized epigenetic modification, many new modifications have been discovered to present in DNA in recent years.  This book provides a comprehensive overview of available techniques and methods together with detailed step-by-step protocols for experimental procedures required to successfully perform analysis on various types of DNA modifications, including 5mC, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 5-carboxycytosine (5caC), 5-hydroxymethyluracil (5hmU), 5-formyluracil (5fU), N6-methyladenine (6mA), β-D-glucosyl-5-hydroxymethyluracil (base J) and 8-oxo-7,8-dihydroguanine (OG). This laboratory manual is a valuable source for biochemists and molecular biologists from different fields who wish to investigate DNA modifications.

This detailed book explores protocols for a wide array of preclinical pharmacology and toxicology evaluations to be applied to chemical drugs and their development through in vitro, involving tissues and cell lines, and in vivo models, using animals as experimental systems, utilized to conduct pharmacological research. Written for the Springer Protocols Handbooks series, the methodologies included in this collection have been standardized by the authors through extensive use in the lab so that they are ready to be applied in the labs of readers around the world. Authoritative and practical, Bioassays in Experimental and Preclinical Pharmacology aims to assist undergraduate and postgraduate students, research scholars, scientists, and other academicians performing research in the vital field of drug discovery.

This detailed book explores protocols for a wide array of preclinical pharmacology and toxicology evaluations to be applied to chemical drugs and their development through in vitro, involving tissues and cell lines, and in vivo models, using animals as experimental systems, utilized to conduct pharmacological research. Written for the Springer Protocols Handbooks series, the methodologies included in this collection have been standardized by the authors through extensive use in the lab so that they are ready to be applied in the labs of readers around the world. Authoritative and practical, Bioassays in Experimental and Preclinical Pharmacology aims to assist undergraduate and postgraduate students, research scholars, scientists, and other academicians performing research in the vital field of drug discovery.

The book provides the readers of various discipline an easy understanding of the latest biophysical techniques pertaining to microbiology. Biofilm associated chronic infection is a major health problem and a serious concern to doctors, scientists and other health workers as it develops antibiotic and multi-drug resistance. This book describes various protocols utilized in the detection of the biofilm. The book has been divided into six sub sections which provides pertinent information about the various biophysical techniques and instruments that are used for detecting and analyzing the biofilm formation upon biotic and abiotic surfaces. The readers will be able to identify the techniques that can best cater information to solve the problem at hand. This book attempts to compile the latest information on the recent advances in the various functional aspects of microbial biofilms, their pathogenesis, present day treatments as well as detection strategies.This book is meant forresearchers in the field of microbiology and interested in understanding microbial pathogenesis, quorum sensing and biofilm formation.

The book provides the readers of various discipline an easy understanding of the latest biophysical techniques pertaining to microbiology. Biofilm associated chronic infection is a major health problem and a serious concern to doctors, scientists and other health workers as it develops antibiotic and multi-drug resistance. This book describes various protocols utilized in the detection of the biofilm. The book has been divided into six sub sections which provides pertinent information about the various biophysical techniques and instruments that are used for detecting and analyzing the biofilm formation upon biotic and abiotic surfaces. The readers will be able to identify the techniques that can best cater information to solve the problem at hand. This book attempts to compile the latest information on the recent advances in the various functional aspects of microbial biofilms, their pathogenesis, present day treatments as well as detection strategies.This book is meant forresearchers in the field of microbiology and interested in understanding microbial pathogenesis, quorum sensing and biofilm formation.