Download and read online Molecular Cloning in PDF and EPUB The first two editions of this manual have been mainstays of molecular biology for nearly twenty years, with an unrivalled reputation for reliability, accuracy, and clarity. In this new edition, authors Joseph 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. Handsomely redesigned and presented in new bindings of proven durability, this three–volume work is essential for everyone using today’s biomolecular techniques. The opening chapters describe essential techniques, some well–established, some new, that are used every day in the best laboratories for isolating, analyzing and cloning DNA molecules, both large and small. These are followed by chapters on cDNA cloning and exon trapping, amplification of DNA, generation and use of nucleic acid probes, mutagenesis, and DNA sequencing. The concluding chapters deal with methods to screen expression libraries, express cloned genes in both prokaryotes and eukaryotic cells, analyze transcripts and proteins, and detect protein–protein interactions. The Appendix is a compendium of reagents, vectors, media, technical suppliers, kits, electronic resources and other essential information. 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.
Download and read online The Condensed Protocols from Molecular Cloning a Laboratory Manual in PDF and EPUB The Condensed Protocols From Molecular Cloning: A Laboratory Manualis a singleâ€“volume adaptation of the threeâ€“volume third edition of Molecular Cloning: A Laboratory Manual.This condensed book contains only the stepâ€“byâ€“step portions of the protocols, accompanied by selected appendices from the world's bestâ€“selling manual of molecular biology techniques. Each protocol is crossâ€“referenced to the appropriate pages in the original manual. This affordable companion volume, designed for bench use, offers individual investigators the opportunity to have their own personal collection of short protocols from the essential Molecular Cloning.
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Download and read online Protein protein Interactions in PDF and EPUB Reflecting the various advances in the field, this book provides comprehensive coverage of protein-protein interactions. It presents a collection of the technical and theoretical issues involved in the study of protein associations, including biophysical approaches. It also offers a collection of computational methods for analyzing interactions.
Download and read online PCR Cloning Protocols in PDF and EPUB Distinguished scientists and researchers present a comprehensive collection of current preparative PCR techniques that can be used in cloning and modifying DNA and cDNA. Topics include performing and optimizing PCR (including long PCR), cloning PCR products, cloning unknown neighboring DNA, and library construction and screening. Also covered are mutagenesis, recombination, and in vitro selection, differential and subtractive approaches to cDNA analysis and screening, and cloning members of gene families. The techniques bring to both new and established researchers the power to apply PCR-based methodology to the cloning and modification of DNA, either through innovative protocols or by fostering individual creativity to modify and customize the protocols to best fit their own needs.
Download and read online Molecular of Cloning of Recombinant Dna in PDF and EPUB Mobilization and Reassembly of Genetic Information documents the proceedings of the Miami Winter Symposium, sponsored by the Department of Biochemistry, University of Miami School of Medicine, Miami, Florida, January 1977. This volume is the 13th in the ""Miami Winter Symposia"" series. Topics for the Miami Winter Symposia focus on areas of biochemistry in which recent progress offers new insights into the molecular basis of biological phenomena. The manuscripts presented by researchers at the symposium cover a wide range of topics including DNA cloning and plasmid biology; yeast DNA expression in Escherichia coli; characterization of tetracycline and ampicillin resistant plasmid cloning vehicles; eukaryotic genome organization; bacterial plasmids containing silk gene sequences; DNA cloning in bacteria for the study of immunoglobulin genes; DNA degradation by rat intestinal nucleases; recombination between bacterial plasmids leading to the formation of plasmid multimers; general methods for inserting specific DNA sequences in cloning vehicles; and cloning and characterization of yeast DNA.
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Download and read online DNA microarrays in PDF and EPUB This manual, designed to extend and to complement the information in the best-selling Molecular Cloning, is a synthesis of the expertise and experience of more than 30 contributors--all innovators in a fast-moving field. DNA Microarrays provides authoritative, detailed instruction on the design, construction, and applications of microarrays, as well as comprehensive descriptions of the software tools and strategies required for analysis of images and data.
Download and read online Methods in Enzymology in PDF and EPUB Requirements for a molecular biology laboratory. General methods for isolating and characterizing nucleic acids. Enzymatic techniques and recombinant DNA technology. Restriction enzymes. Growth and maintenance of bacteria. Genomic cloning. Preparation and characterization of RNA. Preparation of cDNA and the generation of cDNA libraries. Selection of clones from libraries. Identification and characterization of specific clones.
Download and read online Molecular Cloning of Hormone Genes in PDF and EPUB The peptide hormones are small proteins that regulate cellular metabolism through their specific interactions with tissues of the endocrine, nervous, and immune systems, as well as in embry onic development. During the past ten years, refinements in the techniques of recombinant DNA technology have resulted in the cloning of genes encoding approximately 50 different hormonal and regulatory peptides, including those in which the peptides themselves and the mRNAs encoding the peptides are present in only trace amounts in the tissues of origin. In addition to provid ing the coding sequences of recognized hormonal and regulatory peptides, gene sequencing has uncovered new bioactive peptides encoded in the precursor pro hormones that are then liberated along with the hormonal peptides during cellular cleavages of the precursors. The encoding of multiple peptides in a single mono cistronic mRNA appears to be a genetic mechanism for the gener ation of biologic diversification without requiring amplification of gene sequences. Two of the objectives in the assembly of this book are to pre sent, in one volume, the known primary structures of the genes encoding several of the polypeptide hormones and related regulatory peptides, and to provide an account of the various ap proaches that have been used to identify and select the cloned genes encoding these polypeptides. The contents of the two in troductory chapters are intended to provide the reader with a brief background of the approaches to gene cloning and the struc ture and expression of hormone-encoding genes.
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Download and read online Guide to molecular cloning techniques in PDF and EPUB Requirements for a molecular biology laboratory. General methods for isolating and characterizing nucleic acids. Enzymatic techniques and recombinant DNA technology. Restriction enzymes. Growth and maintenance of bacteria. Genomic cloning. Preparation and characterization of RNA. Preparation of cDNA and the generation of cDNA libraries. Selection of clones from libraries. Identification and characterization of specific clones.
Download and read online MOLECULAR CLONING CHARACTERI in PDF and EPUB This dissertation, "Molecular Cloning and Characterization of SNF1 Related Protein Kinases in Tomato (lycopersicon Esculentum)" by Pui-Yi, Lam, 林佩儀, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled MOLECULAR CLONING AND CHARACTERIZATION OF SNF1 RELATED PROTEIN KINASES IN TOMATO (Lycopersicon esculentum) Submitted by Lam Pui Yi for the Degree of Master of Philosophy at The University of Hong Kong in April 2003 Two cDNAs encoding SNF1 related protein kinase (SnRK) were cloned from pink tomato fruit. From the amino acid sequences and phylogenetic analysis, they both fell into the SnRK1 family, and were designated as TKIN1 and TKIN2. The complete cDNA sequence of TKIN1 contains 2045 bp, with the entire coding sequence of 1542 bp that encodes a 514 amino acid protein with a calculated molecular mass of 58824 Da. TKIN1 encodes the protein which has the identical amino acid sequence with tomato SNF1 expressed in seeds imbibed in gibberellin for 24 hours. It also showed high similarity to potato StubSNF1, tobacco NPK5 and cucumber SnRK1. Phylogenetic analysis showed that TKIN1 fell into the SnRK1a sub-family. In contrast, the complete cDNA sequence of TKIN2 contains 1974 bp, with the entire coding sequence of 1512 bp encoding a 504 amino acid protein with a predicted molecular mass of 57762 Da. TKIN2 encodes a protein closely related to potato PKIN1. The phylogenetic tree showed that TKIN2 clusters with potato PKIN1 and lies beyond the SnRK1a and SnRK1b sub-families. Both TKIN1 and TKIN2 contain the activation segment required for activation by the upstream kinase. A comparison of the TKIN1 and TKIN2 amino acid sequences showed that they share overall 67.7% identity, with 87.8% similarity within the kinase domain and 50.0% similarity within the C-terminal regulatory domain. This suggests that these two isoforms are enzymatically similar but are differentially regulated. TKIN1 and TKIN2 cDNAs were heterlogously expressed in E. coli as 6xHis- tagged fusion and thioredoxin fusion proteins to confirm that the cDNAs encode active forms of SnRK. They were successfully expressed as soluble fusion proteins in both systems and the fusion proteins were purified by affinity chromatography. The recombinant proteins however showed no activity in the SAMS peptide phosphorylation assay, an assay specific for the SNF1/AMPK family. It seems that a eukaryotic expression system is required for the expression of biologically active TKIN1 and TKIN2. Attempts were made to determine the relative levels of the TKIN1 and TKIN2 mRNAs by northern blot analysis. However, no distinct signal was detected for either gene. This suggests that the mRNAs encoding TKIN1 and TKIN2 may be expressed at levels too low for detection by northern blots. Polyclonal antibodies were raised against the synthetic peptide VSEESLRRPFRKEKT, corresponding to residues 369-383 of TKIN2. This sequence of amino acids is specific for TKIN2 and fulfills the requirements for raising antibodies. The crude antiserum was pre-adsorbed against tomato tissue proteins and this pre-adsorbed antiserum recognizes a polypeptide with an approximate molecular mass of 57.8 kD in crude extracts of green, breaking, turning, pink, orange and red fruits, on western blot. It showed that TKIN2 protein accumulated in green, breaking and turning fruits and its abundance decreased from pink to red fruits, suggesting that TKIN2 likely plays a role in the tomato fruit ripening process. DOI: 10.5353/th_b2773668 Subjects: Protei
Download and read online Molecular Cloning and Gene Regulation in Bacilli in PDF and EPUB Molecular Cloning and Gene Regulation in Bacilli presents the proceedings of the 1981 Cetus Conference on Genetics held at Stanford University, Stanford, California. It summarizes both basic and applied aspects of bacilli genetics. It discusses significant advances made in understanding chromosome structure, gene arrangement, molecular cloning, cloned gene expression, DNA metabolism, transcription, and translation. Divided into five sessions, the book starts by discussing the DNA sequence from RNA intergenic spaces of Bacillus subtilis rRNA gene sets, the construction of a bifunctional cosmid vector of large DNA segments, and the mating system of bacilli. Molecular cloning session covers complementation system and dominance analyses in Bacillus, genetic fusion of Escherichia coli lac genes to a Bacillus subtilis promoter, and DNA cloning of B. subtilis. It also describes the construction of trimethoprim resistant B. subtilis plasmid and expression of E. coli trp genes cloned in B. subtilis. Session III encompasses chapters that discuss protein secretion by bacilli; regulation of alpha-amylase production in B. subtilis; entomocidal toxin translation of B. thuringiensis; and expression of crystal protein, heterologous, and eukaryotic genes in bacilli. Session IV focuses on various aspects of DNA metabolism of bacilli, such as the interaction of bacterial chromosome with cell membrane; plasmid DNA in competent cells and protoplasts of B. subtilis; analysis of peptides synthesized by B. subtilis mutants; and DNA repair, uptake, restriction, modification, and recombination. The final session examines species-specific translation, control of gene expression and replications in plasmids, development of expression-vector in B. subtilis, and regulatory modifications of RNA polymerase. Each chapter is presented in an experimental manner, consisting of a summary of the study, materials and methods, results, as well as references.
Download and read online Cloning in PDF and EPUB The terms 'recombinant DNA technology', 'DNA cloning', 'molecular cloning' or 'gene cloning' all refer to the same process: the transfer of a DNA fragment of interest from one organism to a self-replicating genetic element such as a bacterial plasmid. The DNA of interest can then be propagated in a foreign host cell. This technology has been around since the 1970s, and it has become a common practice in molecular biology labs today. Reproductive cloning is a technology used to generate an animal that has the same nuclear DNA as another currently or previously existing animal. Dolly was created by reproductive cloning technology. In a process called 'somatic cell nuclear transfer' (SCNT), scientists transfer genetic material from the nucleus of a donor adult cell to an egg whose nucleus, and thus its genetic material, has been removed. The reconstructed egg containing the DNA from a donor cell must be treated with chemicals or electric current in order to stimulate cell division. Once the cloned embryo reaches a suitable stage, it is transferred to the uterus of a female host where it continues to develop until birth. Therapeutic cloning, also called "embryo cloning," is the production of human embryos for use in research. The goal of this process is not to create cloned human beings, but rather to harvest stem cells that can be used to study human development and to treat disease. Stem cells are important to biomedical researchers because they can be used to generate virtually any type of specialised cell in the human body. This new book presents an up-to-date Chronology of Cloning along with current and selected abstracts dealing with cloning as well as a guide to books on the topic. Access to the abstract and books sections is provided by title, subject and author indexes.