| Z718262 Sigma |
Analyzing DNA |
General description
A genome revolution is transforming biomedical research in the leading laboratories. Now, a series of manuals from Cold Spring Harbor puts the tools of the revolution in everyone's hands.
Assuming only a basic knowledge of molecular biology, these manuals explain how to clone, manipulate, analyze, and sequence large segments of DNA, and relate expressed sequence to phenotypic variation.
The techniques are written for application to animal DNA as well as human genomes. They deal plainly with sources of failure - and solutions. Assembled by experienced CSH course instructors, the protocols are written by experts, often the methods' creators, and have been rigorously edited to Cold Spring Harbor standards of accuracy, consistency, and completeness.
A complement to the bible of recombinant DNA, Molecular Cloning, these manuals are essential for every laboratory in which genes are being studied.
Assuming only a basic knowledge of molecular biology, these manuals explain how to clone, manipulate, analyze, and sequence large segments of DNA, and relate expressed sequence to phenotypic variation.
The techniques are written for application to animal DNA as well as human genomes. They deal plainly with sources of failure - and solutions. Assembled by experienced CSH course instructors, the protocols are written by experts, often the methods' creators, and have been rigorously edited to Cold Spring Harbor standards of accuracy, consistency, and completeness.
A complement to the bible of recombinant DNA, Molecular Cloning, these manuals are essential for every laboratory in which genes are being studied.
Table of Contents
Preface
Acknowledgments
Contributors
Abbreviations and Acronyms
1. Purifying and Analyzing Genomic DNA
OVERVIEW OF GENOMIC DNA ANALYSIS< Historical Perspective Issues of Contamination and Safety
ISOLATION OF GENOMIC DNA
DNA FROM MAMMALIAN SOURCES
PROTOCOLS
DNA Isolation from Tissue-culture Cell Lines Growing in Suspension Transforming Lymphoblasts with EBV Maintaining B95-8 and IA-3 EBV-producing Cell Lines
DNA Isolation from Adherent Fibroblasts Using Trypsin
DNA Isolation from Adherent Fibroblasts ("Sliming")
DNA Isolation from Peripheral Blood DNA Isolation from Fresh and Frozen Tissues Isolation from a Mammalian Model Organism: The Mouse
DNA FROM ANIMAL MODEL ORGANISMS
PROTOCOLS
DNA Isolation from D. melanogaster
DNA Isolation from C. elegans
DNA FROM PLANTS
PROTOCOL
DNA Isolation from Arabidopsis thaliana
DNA FROM YEAST
PROTOCOLS
DNA Isolation from S. cerevisiae
DNA Isolation from S. pombe
DNA FROM BACTERIA
PROTOCOL
DNA Isolation from E. coli
TROUBLESHOOTING FOR GENOMIC DNA ISOLATION
Degradation and Fragmentation of the DNA Poor Recovery of DNA
Contamination of the DNA with Proteins
Contamination of the DNA with RNA
SOUTHERN BLOTTING
DIGESTION OF DNA WITH RESTRICTION ENZYMES
PROTOCOL Digesting Genomic DNA with Restriction Enzymes
ELECTROPHORETIC SEPARATION OF DIGESTION PRODUCTS
PROTOCOL Size Fractionating Digested DNA on Agarose Gels
TRANSFER OF DNA ONTO SOLID SUPPORTS
PROTOCOL Transferring Size-fractionated DNA onto Solid Supports
HYBRIDIZATION OF PROBES TO IMMOBILIZED DNA
PROTOCOL Gel Purification of DNA Fragments for Use as Probes
RADIOLABELING DNA PROBES
PROTOCOLS Radiolabeling Probes by Random Priming with Hexameric Oligonucleotides End-labeling Oligonucleotide Probes
BLOCKING REPETITIVE DNA SEQUENCES
PROTOCOLS Preannealing Probes to Cot 1 Blocking DNA Preannealing Probes to Genomic Blocking DNA
DETECTING UNIQUE SEQUENCES ON MEMBRANES BY USING RADIOLABELED PROBES PROTOCOLS Hybridization Using Probes Labeled by Random Priming Hybridization Using End-labeled Oligonucleotide Probes
USE OF NONRADIOACTIVE PROBES FOR HYBRIDIZATION Biotinylation of Probe DNA Membrane Preparation Hybridization and Washing Detection of Chemiluminescence
TROUBLESHOOTING FOR SOUTHERN BLOTTING
Problems with the DNA Sample Problems with the Gel Problems with Transferring the DNA onto the Solid Support Problems with Hybridizing Radioactive or Nonradioactive Probes to the DNA.
Acknowledgments
Contributors
Abbreviations and Acronyms
1. Purifying and Analyzing Genomic DNA
OVERVIEW OF GENOMIC DNA ANALYSIS< Historical Perspective Issues of Contamination and Safety
ISOLATION OF GENOMIC DNA
DNA FROM MAMMALIAN SOURCES
PROTOCOLS
DNA Isolation from Tissue-culture Cell Lines Growing in Suspension Transforming Lymphoblasts with EBV Maintaining B95-8 and IA-3 EBV-producing Cell Lines
DNA Isolation from Adherent Fibroblasts Using Trypsin
DNA Isolation from Adherent Fibroblasts ("Sliming")
DNA Isolation from Peripheral Blood DNA Isolation from Fresh and Frozen Tissues Isolation from a Mammalian Model Organism: The Mouse
DNA FROM ANIMAL MODEL ORGANISMS
PROTOCOLS
DNA Isolation from D. melanogaster
DNA Isolation from C. elegans
DNA FROM PLANTS
PROTOCOL
DNA Isolation from Arabidopsis thaliana
DNA FROM YEAST
PROTOCOLS
DNA Isolation from S. cerevisiae
DNA Isolation from S. pombe
DNA FROM BACTERIA
PROTOCOL
DNA Isolation from E. coli
TROUBLESHOOTING FOR GENOMIC DNA ISOLATION
Degradation and Fragmentation of the DNA Poor Recovery of DNA
Contamination of the DNA with Proteins
Contamination of the DNA with RNA
SOUTHERN BLOTTING
DIGESTION OF DNA WITH RESTRICTION ENZYMES
PROTOCOL Digesting Genomic DNA with Restriction Enzymes
ELECTROPHORETIC SEPARATION OF DIGESTION PRODUCTS
PROTOCOL Size Fractionating Digested DNA on Agarose Gels
TRANSFER OF DNA ONTO SOLID SUPPORTS
PROTOCOL Transferring Size-fractionated DNA onto Solid Supports
HYBRIDIZATION OF PROBES TO IMMOBILIZED DNA
PROTOCOL Gel Purification of DNA Fragments for Use as Probes
RADIOLABELING DNA PROBES
PROTOCOLS Radiolabeling Probes by Random Priming with Hexameric Oligonucleotides End-labeling Oligonucleotide Probes
BLOCKING REPETITIVE DNA SEQUENCES
PROTOCOLS Preannealing Probes to Cot 1 Blocking DNA Preannealing Probes to Genomic Blocking DNA
DETECTING UNIQUE SEQUENCES ON MEMBRANES BY USING RADIOLABELED PROBES PROTOCOLS Hybridization Using Probes Labeled by Random Priming Hybridization Using End-labeled Oligonucleotide Probes
USE OF NONRADIOACTIVE PROBES FOR HYBRIDIZATION Biotinylation of Probe DNA Membrane Preparation Hybridization and Washing Detection of Chemiluminescence
TROUBLESHOOTING FOR SOUTHERN BLOTTING
Problems with the DNA Sample Problems with the Gel Problems with Transferring the DNA onto the Solid Support Problems with Hybridizing Radioactive or Nonradioactive Probes to the DNA.
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